Journal Pre-proof

Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores

with supplementation of medicinal plant extracts: Development of a programmable
controlled-rate method and a practical method

Subuntith Nimrat, Supattra Noppakun, Kanokon Sripuak, Traimat Boonthai, Verapong

Vuthiphandchai
PII: S0044-8486(19)31682-5
DOI: https://doi.org/10.1016/j.aquaculture.2019.734537
Reference: AQUA 734537

To appear in: Aquaculture

Received Date: 4 July 2019

Revised Date: 8 September 2019
Accepted Date: 23 September 2019

Please cite this article as: Nimrat, S., Noppakun, S., Sripuak, K., Boonthai, T., Vuthiphandchai,
V., Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores with
supplementation of medicinal plant extracts: Development of a programmable controlled-rate method
and a practical method, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734537.

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1 Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores with

2 supplementation of medicinal plant extracts: development of a programmable controlled-rate

3 method and a practical method

6 Subuntith Nimrata*, Supattra Noppakunb, Kanokon Sripuakb, Traimat Boonthaic

7 and Verapong Vuthiphandchaib

10

a
11 Department of Microbiology and Environmental Science Program, Faculty of Science, Burapha

12 University, Chon Buri 20131, Thailand

b
13 Department of Aquatic Science, Faculty of Science, Burapha University,

14 Chon Buri 20131, Thailand

c
15 Biological Science Program, Faculty of Science, Burapha University,

16 Chon Buri 20131, Thailand

17

18

19

20 *Corresponding author. Tel. +66 38-103-120; Fax: +66 38-393-490

21 E-mail address: subunti@buu.ac.th (S. Nimrat); supattra.nop@cpmail.in.th (S. Noppakun);

22 Eay_14@hotmail.com (K. Sripuak); boonthait@gmail.com (T. Boonthai);

23 verapong@buu.ac.th (V. Vuthiphandchai)

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24

25

26 ORCID

27 Dr. Subuntith Nimrat: https://orcid.org/0000-0001-9327-2408

28 Dr. Traimat Boonthai: https://orcid.org/0000-0002-8251-6486

29 Dr. Verapong Vuthiphandchai: https://orcid.org/0000-0003-3311-4939

30

31 Abstract

32 The objectives of this study were to generate the optimal cryopreservation protocols

33 (programmable controlled-rate and practical methods) for banana shrimp (Fenneropenaeus

34 merguiensis) spermatophores and investigate the efficacy of plant extracts (moringa, Moringa

35 oleifera Lam and ginger, Zingiber officinale Roscoe) on sperm survival and bacterial abundance

36 in cryostored spermatophores. Spermatophores were exposed to various cryoprotectants at

37 different concentrations and equilibrium times prior to examining sperm viability. A minimal

38 toxicity to banana shrimp sperm was observed in spermatophores suspended in 5% DMSO. The

39 optimal freezing protocol using a programmable controlled-rate freezer included exposure of

40 spermatophores in calcium-free saline (Ca-F saline) containing 5% DMSO with 20 min

41 equilibration time, and then utilization of a one-step freezing at the rate of -2 °C min-1 from 25

42 °C to -80 °C and holding for 30 s before storage in a liquid nitrogen tank. Highest sperm viability

43 of post-thawed spermatophores was obtained after thawing at 30 °C for 2 min in a water bath.

44 Cryopreservation of F. merguiensis spermatophores using a practical method was achieved by

45 suspending spermatophores in Ca-F saline containing 5% DMSO with equilibration time of 20

46 min at 25 °C and placing at 4 cm over liquid nitrogen surface (a freezing rate of about -2.5 °C
 3

47 min-1) prior to plunging into liquid nitrogen. In order to evaluate the efficacy of plant extracts on

48 sperm survival and bacterial abundance of cryostored spermatophores, two freezing protocols

49 (programmable and practical methods) with ethanolic moringa and ginger extracts (0.1 mg mL-1

50 each) were developed for long-term cryostorage. Moringa and ginger extracts had no detrimental

51 effect on F. merguiensis sperm due to retaining high viable sperm without significant differences

52 (P > 0.05) compared to the control after a 12-month cryostorage. Supplementation of moringa or

53 ginger extract into sperm extender resulted in a reduction of bacterial abundance in cryostored

54 spermatophores. This report, for the first time, indicates the promising potential of plant extracts

55 as an alternative method for controlling bacterial contaminants in cryopreserved spermatophores

56 of penaeid shrimp.

57

58 Keywords: Fenneropenaeus merguiensis; Spermatophore; Cryopreservation; Moringa; Ginger;

59 Bacteria

60

61 Highlights of the manuscript

62 - This study reports, for the first time, the cryopreservation protocol using a

63 programmable controlled-rate freezer for banana shrimp (Fenneropenaeus merguiensis)

64 spermatophores.

65 - This study also develops a practical method for cryopreservation of banana shrimp

66 spermatophores using a Styrofoam box, which would provide the simpler and more affordable

67 cryopreservation protocol for hatchery operations.

68 - This study, for the first time, reports the potential use of extracts from moringa

69 (Moringa oleifera Lam.) and ginger (Zingiber officinale Roscoe) as alternative antimicrobial
 4

70 agents for controlling bacterial contaminants in cryopreserved spermatophores of penaeid

71 shrimp to reduce the use of harmful pharmaceuticals in aquaculture and provide a more

72 reliable supply of spermatophores without a biosafety concern.

73

74 1. Introduction

75 Shrimp culture is economically important in Thailand with an annual production of

76 378,300 tons in 2016 reaching a market value of approximately 1,875 million US$ (Fisheries

77 Statistics of Thailand, 2018). Currently, viral and bacterial diseases have posed a serious threat to

78 shrimp culture industry. One such example is Acute Hepatopancreatic Necrosis Disease,

79 commonly referred to as Early Mortality Syndrome associated with Vibrio parahaemolyticus

80 infection, which caused economic losses of nearly 800 million US$ in Thailand. This was

81 primarily due to 41.8%reduction in whiteleg shrimp (Litopenaeus vannamei) production from

82 2011 to 2014 (Fisheries Statistics of Thailand, 2016). Because of the disease outbreaks in shrimp

83 farming, similar to, black tiger (Penaeus monodon) and whiteleg shrimp, banana shrimp

84 (Fenneropenaeus merguiensis), has the potential to become an alternative commercial species

85 for shrimp domestication and breeding programs in Thailand. However, since production of

86 banana shrimp is still largely dependent on wild seeds and broods, which can be unpredictable in

87 terms of the quality and quantity owing to the habitat deterioration and overharvest, availability

88 of quality seed for commercial production has been problematic. Hatchery production of banana

89 shrimp seed is necessary if the shrimp culture is to meet the market needs.

90 Cryopreservation plays an important role in development of reproductive technologies

91 because of allowing long-term preservation of genetic materials from rare or valuable breeds,

92 securing genetics for future use and enabling gamete to be easily marketed and transported
 5

93 (Herraez et al., 2012). To date, reliable protocols of spermatophore cryopreservation were

94 reported in a number of decapod crustaceans, e.g. Ridgeback rock shrimp, Sicyonia ingentis

95 (Anchordoguy et al., 1988), giant freshwater shrimp, Macrobrachium rosenbergii (Chow et al.,

96 1985; Akarasanon et al., 2004; Valentina claudet et al., 2016), Indian white shrimp,

97 Fenneropenaeus indicus (Diwan and Joseph, 1999; Selvakumar et al., 2018), fleshy shrimp,

98 Penaeus chinensis (Ke and Cai, 1996), black tiger shrimp (Bart et al., 2006; Vuthiphandchai et

99 al., 2007), and whiteleg shrimp (Lezcano et al., 2004; Chao et al., 2009). Recently,

100 cryopreservation of banana shrimp spermatophore was developed by Memon et al. (2012), who

101 focused on designing the protocol successful in terms of optimal sperm extender, cryoprotectant,

102 freezing and thawing rates using a household and laboratory instrument. Moreover, most

103 successful attempts to cryostore shrimp spermatophores/sperm have focused on identifying an

104 optimal freezing rate designed using a sophisticated controlled-programmable freezer. In spite of

105 a practical method successfully applied in sperm cryopreservation of various fish and oyster

106 species (Adams et al., 2004; Irawan et al., 2010), little is known how shrimp spermatophores are

107 cryostored under the suitable condition using a simple freezing protocol. Therefore, this

108 technique needs to be developed to gain the simpler and more affordable cryopreservation for

109 hatchery operations.

110 Despite spermatophore cryopreservation capable of maintaining high sperm quality,

111 sperm cryopreservation has encountered a serious issue regarding biosafety due to contamination

112 of potential pathogen (Boonthai et al., 2016a). In general, bacteria contaminate easily in

113 spermatophores during related steps of cryopreservation, e.g. spermatophore collection, extender

114 and cryoprotectant preparation, processing and storage, resulting in the presence of viable

115 bacteria during cryostorage of sperm. A divergence of bacterial flora was reported in
 6

116 spermatophores of black tiger and whiteleg shrimp, especially pathogenic and spoilage bacteria

117 (Nimrat et al., 2005, 2006, 2008; Ueno et al., 2015). This has put forward to generate a novel

118 technology to decontaminate pathogens while maintaining desirable sperm quality. In spite of

119 antibiotics having broad spectrum of bactericidal activity without harmful effects on shrimp

120 sperm (Nimrat et al., 2005, 2006), use of these pharmaceuticals have resulted in emergence and

121 dissemination of antibiotic resistant bacteria and resistance genes into environments (Cabello et

122 al., 2016).

123 Supplementation of medicinal plant extract as an antimicrobial agent in sperm extender is

124 a complementary technology due to its environmental-friendliness, which would provide a more

125 reliable supply of spermatophores without a biosafety concern. Medicinal plants are generally

126 used in aquaculture as an alternative to chemotherapy. For example, moringa (Moringa

127 oleifera Lam.) leaf powder has the potential to control bacterial diseases caused by Vibrio

128 harveyi because of dramatically improved survival in infected Indian white shrimp (Rayes,

129 2013). Dried powder of ginger (Zingiber officinale Roscoe) facilitates nutrient assimilations and

130 immunomodulatory functions in Sobaity sea bream (Sparidentex hasta) of which a marked

131 increase in growth and protection against pathogenic Photobacterium damselae was conferred

132 when the ginger administrated in feed (Jahanjoo et al., 2018). To date, there is no available report

133 focused on application of medicinal plant extracts in sperm extender for cryopreservation of

134 penaeid spermatophores. Therefore, the aims of this study were to develop the cryopreservation

135 protocols (a programmable controlled-rate freezer method and a simple freezing method) with

136 plant extract supplements, moringa and ginger, for long-term cryostorage of banana shrimp

137 spermatophores in liquid nitrogen, and to evaluate the efficacy of plant extracts on sperm

138 survival and bacterial abundance in cryostored spermatophores.

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139

140 2. Materials and methods

141 2.1 Broodstock and spermatophore collection

142 Sexually mature F. merguiensis males were caught from the natural spawning grounds

143 around Si Chang Islands and its vicinity in the Gulf of Thailand, Chon Buri Province by a local

144 supplier. Healthy males with intact appendages and normal physical conditions were transported

145 to a commercial hatchery within 6 h post-capture. Upon arrival, all males were maintained in 15-

146 m2 rectangular tanks with a cartridge-filtered seawater (30‰) depth of about 90 cm, at 27-28.5

147 °C. Broodstocks were stocked at a density of 10 individuals per m2. Shrimp were fed twice a day

148 with mixed fresh squid and polychaetes (approximately 10% body weight/day) by dividing into

149 two equal portions for the morning and evening feedings. Uneaten food and debris were removed

150 2 h post-feeding. Water exchange was done daily at approximately 40-50%. Broodstocks were

151 maintained under a natural photoperiod of approximately 12 h light and 12 h dark. Shrimp were

152 held for 3 d before beginning of the experiment to reduce the stress from capture. Average

153 weight and length of males (N = 550) were 27.8 ± 6.2 g and 15.2 ± 3.4 cm, respectively.

154 Mature male F. merguiensis with an opaque white swelling around the coxae at the base

155 of the fifth walking leg (pereiopods) were rinsed with sterile seawater. The gonopores were then

156 sprayed with 70% ethanol to eliminate external contaminants prior to collection of

157 spermatophores. Manual ejaculation of spermatophores was applied by gentle pressure with the

158 thumb and forefinger between the abdomen and the base of the fifth walking leg. The extruded

159 spermatophores were gently collected using the sterile forceps. A small drop of povidone-iodine

160 was applied as antiseptic to the genital area before releasing the males back into the tank. Two

161 spermatophores were obtained from each male. Only non-melanized spermatophores were
 8

162 selected for the experiments. During spermatophore collection, the sanitation procedures with

163 aseptic techniques for sperm cryopreservation set by Boonthai et al. (2016a) were implemented

164 in this study. Collected spermatophores were then kept in sterile petri dishes containing Ca-F

165 saline in an ice box and brought to the cryopreservation laboratory, Faculty of Science, Burapha

166 University, for preservation studies within 15 min.

167

168 2.2 Effect of cryoprotectant exposure on sperm survival

169 To evaluate effect of cryoprotectants on sperm viability, six cryoprotectants were selected

170 in this study, namely, dimethyl sulfoxide (DMSO), sucrose, 1,2-propylene glycol (PG), methanol

171 (MeOH), formamide (FM), and acetamide (Ace). A 6 x 4 x 6 factorial design (six

172 cryoprotectants, four concentrations and six equilibration times) in completely randomized trial

173 was established in four replicates at an ambient temperature (25 °C). Each cryoprotectant

174 solution was prepared to produce a final concentration of 5, 10, 15 and 20% (v v-1) with sterile

175 calcium-free saline (Ca-F saline). Ca-F saline was used as a sperm extender in this study because

176 of its retaining a high viability of F. merguiensis sperm during a 90-d cryostorage of

177 spermatophore (Memon et al., 2012), which was also in accordance with our preliminary study.

178 The Ca-F saline was composed of (g L-1): 21.63 NaCl, 1.12 KCl, 0.53 H3BO3, 0.19 NaOH and

179 4.93 MgSO4⋅7H2O in sterile distilled water (adjusted pH to 7.4 with 1 N HCl. This extender was

180 passed through a 0.45 µm membrane filter (Sartorius, Bedford, Massachusetts, USA) to

181 eliminate bacterial contaminants. All chemicals were purchased from Sigma Chemicals

182 Company (St. Louis, Missouri, USA). Each spermatophore was individually placed in a 1.5-mL

183 cryovial (Nalgene, Rochester, New York, USA) and then, a cryoprotectant solution (1 mL) at

184 each concentration was gradually added into the cryovial to reduce sudden shock due to osmotic
 9

185 change. Freshly collected spermatophores suspended in Ca-F saline without cryoprotectant were

186 used and considered as the controls. During the exposure periods at the room temperature (25

187 °C), the vials were not lidded to allow oxygen penetration. Viable sperm assessment was

188 performed at the beginning of experiment (0), 10, 20, 30, 45 and 60 min post-exposure.

189 Spermatophores were rinsed twice with sterile Ca-F saline to remove cryoprotectant residues and

190 then prepared to estimate sperm viability (see below). Each treatment had four replicates of

191 assessment.

192

193 2.3 Development of the freezing protocols

194 Sucrose (5%) and DMSO (5%) with a 20-min equilibration period were selected for

195 cryopreservation studies of spermatophores because of retaining high sperm viability. In our

196 preliminary study, spermatophores cryostored using the freezing rates of -1, -2, -3 and -4 °C min-

197
1
from 25 °C to -30 °C before plunging into liquid nitrogen exhibited low sperm viability.

198 Therefore, the use of one-step freezing regime from an initial temperature of 25 °C to a final

199 temperature of -80 °C with different freezing rates was investigated in this study. A combination

200 of 5% DMSO with 5% sucrose was also incorporated for cryopreservation study.

201 Spermatophores (N = 144) were suspended in sterile Ca-F saline (10 mL) at an ambient

202 temperature (25 °C) for 5 min prior to transferring into a 1.5-mL cryovial (one spermatophore

203 per vial). Afterwards, a small amount of 5% DMSO or 5% sucrose in Ca-F saline (1 mL) was

204 added into the cryovials. To study the effects of a dual cryoprotectant on cryopreservation of F.

205 merguiensis spermatophores, treatments associated with simultaneous addition of 5% sucrose

206 and 5% DMSO or addition of 5% sucrose, and then 5% DMSO (0.5 mL each) into the vials

207 containing spermatophores were setup. After 20 min equilibration period at the ambient
 10

208 temperature, half of cryoprotectant solutions in the vials were decanted. Cryovials were then

209 capped and placed in the cooling chamber of a programmable controlled-rate freezer (model

210 3000, Cryologic Pty., Blackburn, Victoria, Australia). Spermatophore cryopreservation was

211 carried out using a one-step freezing with the cooling rates of -1, -2, -3 and -4 °C min-1 from 25

212 °C to a final temperature of -80 °C and, then holding for 30 s before storing in a liquid nitrogen

213 tank (Model XT-34, Taylor-Wharton, Theodore, Alabama, USA). Apparent sperm viability was

214 examined at 5 min and 1 h post-cryostorage. After thawing in a water bath at 30 °C for 2 min,

215 post-thawed spermatophores were rinsed rapidly twice with sterile Ca-F saline and, then

216 immediately evaluated for sperm viability (see below). Cryopreservation experiments were done

217 in four replicates. Spermatophores suspended in Ca-F saline without cryoprotectant were served

218 as the controls.

219

220 2.4 Thawing temperature study

221 To ascertain the optimal thawing temperature, spermatophores cryopreserved using the

222 most effective protocol, regarded as addition of 5% DMSO in Ca-F saline before freezing at a

223 rate of 2 °C min-1 between 25 °C and -80 °C, were stored in the liquid nitrogen tank for 24 h.

224 Afterwards, frozen spermatophores were thawed at four different temperatures (30, 50, 70 and 90

225 °C for 2 min each) in a water bath with four replicates. Post-thawed spermatophores were rinsed

226 rapidly twice with sterile Ca-F saline prior to evaluating viable sperm immediately (see below).

227 Percentage of sperm viability of freshly collected spermatophores was used as the control.

228

229 2.5 Plant procurement and extraction

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230 Moringa and ginger extracts were selected in this study owing to their abilities to

231 maintain viable sperm (> 70%) and inhibit growth of pathogenic Vibrio and Pseudomonas in

232 chilled F. merguiensis spermatophores during a 14-d storage. Herbs were field-collected in a

233 local botanical garden in Chon Buri Province, and identified by a Thai herbalist. Rinsed moringa

234 leaves and ginger rhizomes were cut into small pieces and dried in a plant drier at 35 °C for 72 h.

235 Dry herb materials were ground using a fine grinder and kept in a zip-lock plastic bag in a

236 desiccator chamber before being used.

237 Each ground herb was soaked in 95% denatured EtOH at ratio of 1 g herb powder: 10 mL

238 ethanol. An Erlenmeyer flask containing plant suspension was agitated in an incubator shaker

239 (New Brunswick Innova 4340, Edison, New Jersey, USA) at 120 rpm, 30 °C for 72 h (Quave et

240 al., 2008). Herb suspension was vacuum-filtered through Whatman no. 1 paper and evaporated at

241 40 °C using a rotary evaporator (Buchi R-205/V, Flawil, Switzerland). Ethanolic extracts were

242 dissolved in DMSO before use.

243

244 2.6 Cryopreservation protocol with supplementation of medicinal plant extracts for long-

245 term storage of spermatophores using a programmable controlled-rate freezer

246 Ca-F saline and Ca-F saline containing 5% DMSO were prepared prior to experiment

247 start. Spermatophores (N = 174) collected from sexually mature males were placed in a 1.5-mL

248 cryovial containing sterile Ca-F saline (10 mL) at 25 °C for 5 min. Weight of each

249 spermatophore was measured and labeled on the side of a 1.5-mL cryovial, then each

250 spermatophore was transferred individually into the cryovial. Cryogenic vial containing one

251 spermatophore was gradually added with 1 mL of Ca-F saline containing 5% DMSO (the

252 control) or Ca-F saline containing 5% DMSO with one of these supplements: penicillin-
 12

253 streptomycin (PS; 10,000 units mL-1 penicillin and 10 g L-1 streptomycin, Invitrogen, Grand

254 Island, New York, USA), moringa extract, and ginger extract at a final concentration of 0.1%

255 (w/v) each. Supplementation of 0.1% PS in sperm extender resulted in a minimal reduction in

256 sperm viability (>78%) and an effective elimination of potential pathogenic Vibrio and

257 Aeromonas in F. merguiensis spermatophores during a 6-month cryostorage and was also

258 favorable for chilled-stored spermatophores of black tiger and whiteleg shrimp (Nimrat et al.,

259 2005, 2006). After 20 min equilibration at the room temperature, half of cryoprotectant solution

260 was removed from the cryovials. Spermatophores were cooled using a one-step freezing at -2 °C

261 min-1 from 25 °C to -80 °C, held for 30 s and then immersed in liquid nitrogen. Six replicates for

262 each treatment were done. Cryopreserved spermatophores were stored in a liquid nitrogen tank

263 for up to 12 months. Apparent viable sperm and bacterial profile were monitored at 5 min post-

264 plunging in liquid nitrogen and 15-min, 1, 3, 6, 9 and 12-month post-cryostorage (see below).

265 Frozen spermatophores were thawed at 30 °C for 2 min in a water bath. At the beginning of the

266 experiment, six freshly collected spermatophores were evaluated for baseline data of sperm

267 viability and bacterial abundance.

268

269 2.7 Cryopreservation protocol with plant extract supplements for long-term storage of

270 spermatophores using a simple freezing method

271 A simple cryopreservation protocol for F. merguiensis spermatophores was developed in

272 our laboratory. Spermatophores (N = 174) were suspended in sterile Ca-F saline (10 mL) at an

273 ambient temperature (25 °C) for 5 min prior to placing into a 1.5-mL cryovial (one

274 spermatophore per vial). The experimental design was divided into four respective treatments as

275 described earlier. Spermatophores were equilibrated in cryoprotectant solution for 20 min at 25
 13

276 °C and, then cryoprotectant solution was partially removed from the vials prior to freezing.

277 Cryocanes with the cryogenic vials were horizontally placed on a rectangular frame (rack),

278 which was 4-cm above liquid nitrogen surface in a Styrofoam box (a dimension of 17 cm width x

279 32 cm length x 20 cm height). Temperature change was monitored by inserting a K-type

280 thermocouple probe (HI 91530K, Hanna Instruments Inc., Rhode Island, USA) into the cryovial.

281 The cryovials containing spermatophores were frozen in liquid nitrogen vapor for 10 min.

282 Cooling rate used in this study was approximately -2.5 °C min-1. Then, all cryogenic vials were

283 plunged into liquid nitrogen and stored in a liquid nitrogen tank. Frozen spermatophores were

284 randomly sampled at 5-min post-plunging the cryovials into liquid nitrogen as well as 15 min, 1,

285 3, 6, 9 and 12-month post-cryostorage. After thawing in a water bath at 30 °C for 2 min, post-

286 thawed spermatophores were prepared for evaluation of apparent viable sperm and bacterial

287 abundance as described below.

288

289 2.8 Sperm viability assessment

290 Evaluation of sperm viability was carried out using a modified eosin-nigrosin exclusion

291 technique (Vuthiphandchai et al., 2007; Nimrat et al., 2008). Freshly collected and post-thawed

292 spermatophores were 100-fold diluted with sterile Ca-F saline and homogenized manually in a

293 glass tissue grinder to form a sperm suspension. A small volume of sperm suspension (50 µL)

294 was mixed with 50 µL of 0.5% eosin and 10% nigrosin solution and then, air-dried on a glass

295 slide. Sperm viability was determined at 100x magnification using a light microscope (Zeiss

296 model D-7085, Berlin, Germany) in six replicates for each treatment. Live sperm was unstained

297 against dark pink-purple background of nigrosin while dead sperm appeared pink-purple. Viable

298 sperm was expressed as percentage, by counting a minimum number of 200 sperm cells per
 14

299 slide, in order to assess sperm survival exposed to cryoprotectants and post-thawed sperm

300 viability of cryopreserved spermatophores subjected to various treatments.

301

302 2.9 Bacteriological study of spermatophores during a 12-month cryostorage

303 Abundance of culturable heterotrophic bacteria (CHB) and culturable Vibrio was

304 evaluated at 5 min post-plunging in liquid nitrogen and 15-min, 1, 3, 6, 9 and 12-month post-

305 cryostorage in a liquid nitrogen tank using a spread plating technique (Nimrat et al., 2006). A

306 100-fold diluted sperm suspension was serially diluted with physiological saline. An aliquot (0.1

307 mL) was spread-plated onto Plate Count Agar (BD Biosciences, Sparks, Maryland, USA) and

308 Thiosulfate Citrate Bile-salts Sucrose agar (BD Biosciences) to enumerate CHB and culturable

309 Vibrio, respectively. After incubation at 30 ± 1 °C for 24-48 h, all colonies grown on the media

310 were counted and calculated as colony forming unit (CFU) g-1. Identification of bacterial species

311 was made following a classical protocol (Holt et al., 1994) and API test kits (bioMerieux, Marcy

312 I’ Etoile, France) following manufacturer’s instruction. Bacterial enumeration was done in

313 triplicates throughout the experiments.

314

315 2.10 Data analysis

316 All data were expressed as mean ± standard deviation. Normality of the data was tested

317 prior to analysis performed. Percentages of sperm viability were arcsine-transformed while

318 bacterial numbers were log-transformed before comparison analysis. Differences among

319 treatments were tested using a Two-way analysis of variance (ANOVA) with Duncan’s multiple

320 range test applied to identify any difference of parameters at a significant level of P < 0.05. A

321 one-way ANOVA was used to find significant difference of sperm viability among
 15

322 spermatophores thawed at various temperatures. All statistical analyses were performed using the

323 Statistical Package for the Social Sciences software (SPSS version 19.0, Chicago, Illinois, USA).

324

325 3. Results

326 3.1 Cryoprotectant effect on sperm viability

327 DMSO exhibited low lethal effect to the sperm at low concentrations (5% and 10%; Fig

328 1). Viable sperm of spermatophores exposed to 5% and 10% DMSO at the beginning of the

329 experiment were not significantly different (P > 0.05) with average values of 75.6 ± 2.1% and

330 85.3 ± 1.6%, respectively. However, sperm viability significantly (P < 0.05) decreased to 57.5 ±

331 3.3% when spermatophores were suspended in 5% DMSO for 30 min while exposure to 10%

332 DMSO for 20 min resulted in a significant (P < 0.05) reduction in sperm survival (58.5 ± 2.2%).

333 High toxicities of DMSO were observed by 20 min post-exposure, when spermatophores were

334 suspended in DMSO at high concentrations (15 - 20%) due to the presence of less than 50%

335 viable sperm. Sperm viability was less than 30% in 20% DMSO by 10 min post-exposure.

336 Sucrose had a moderate toxicity to sperm at low concentrations (5% and 10%) while

337 MeOH seemed to be moderately toxic at all concentrations tested. Sperm viability of

338 spermatophoes subjected with sucrose decreased significantly (P < 0.05) inversely with

339 concentration and exposure time. A significant reduction (P < 0.05) in sperm survival was

340 observed when spermatophores exposed to either 5% sucrose for 30 min from 60.9 ± 1.7% to

341 42.2 ± 1.6%, or 10% sucrose for 20 min from 72.1 ± 1.5% to 51.7 ± 1.3% (Fig. 1). Exposure to

342 MeOH at various concentrations produced a relatively variable survival of sperm.

343 Spermatophores immersed in MeOH at all concentrations (5-20%) at the beginning of the

344 experiment had similar sperm viability in the ranges of 78.6 ± 2.5% to 89.6 ± 2.2%. Sperm
 16

345 viabilities dropped to 52.0 ± 6.1% after 20 min exposure to 5% MeOH and to 45.9 ± 4.0% and

346 38.5 ± 2.0% after 10 min exposure to 10% and 15% MeOH, respectively (Fig. 1).

347 Other cryoprotectants demonstrated different degree of toxicity to F. merguiensis sperm

348 by which PG, FM and Ace were severely toxic. Although viable sperm (>70%) were observed at

349 the beginning of the experiment, except Ace at 5-15%, an increase in exposure period to 10, 20,

350 30, 45 and 60 min decreased significantly (P < 0.05) the percentage of viable sperm at all

351 concentrations (Fig. 1). Spermatophores suspended in 20% PG, 20% FM and 15-20% Ace

352 exhibited complete mortality of sperm by 30, 20 and 10-20 min of exposure, respectively.

353

354 3.2 Freezing protocol

355 The highest post-thawed sperm viability (89.9 ± 2.5%) was observed from a treatment

356 using 5% DMSO and a freezing rate of -2 °C min-1, not significantly different (P > 0.05) from a

357 treatment added 5% sucrose and 5% DMSO simultaneously and frozen at -2 °C min-1 (82.9 ±

358 3.7%; Table 1) at 5 min post-cryostorage. After 1-h cryostorage, sperm viability of

359 spermatophores frozen with 5% DMSO and -2 °C min-1 was 83.5 ± 2.7%, significantly (P <

360 0.05) higher than that of spermatophores simultaneously added with 5% sucrose and 5% DMSO

361 prior to freezing at -2 °C min-1 (61.4 ± 1.2%). Cryopreservation of F. merguiensis

362 spermatophores using higher freezing rates (-3 °C min-1 and -4 °C min-1) resulted in lower sperm

363 survivals regardless of cryoprotectants used (Table 1).

364

365 3.3 Thawing temperatures

366 Percentage of viable sperm decreased significantly (P < 0.05) at higher thawing

367 temperatures (Table 2). Freshly collected spermatophores of F. merguiensis had an average
 17

368 sperm viability of about 88.4 ± 0.8%. Highest percentage of viable sperm of cryopreserved

369 spermatophores (85.1 ± 1.4%) was observed after thawing at 30 °C for 2 min (Table 2) by which

370 intact structure of sperm remained unstained (Fig. 2). Cryopreserved spermatophores thawed at

371 50 °C for 2 min exhibited post-thawed sperm viability of 61.6 ± 0.8%, which was significantly

372 higher than that at 70 °C for 2 min (25.3 ± 1.2%) and 90 °C for 2 min (15.8 ± 1.8%). The use of

373 high thawing rates (70 and 90 °C for 2 min each) resulted in the incidence of membrane rupture

374 of F. merguiensis sperm (Fig. 2; inset), and consequently low post-thawed sperm viability.

375

376 3.4 Effect of medicinal plant extracts on sperm viability

377 When using a programmable controlled-rate freezer, sperm viability of post-thawed F.

378 merguiensis spermatophores did not differ significantly (P > 0.05) among treatments during a

379 12-month cryostorage period (Fig. 3). Sperm viability of freshly collected spermatophores was

380 99.8 ± 0.2%. Similar sperm viabilities between the control and spermatophores treated either

381 with 0.1% PS, moringa or ginger extract were observed in cryopreserved spermatophores

382 cryostored for 12 months with average values of 99.1 ± 0.5%, 98.4 ± 1.6%, 99.1 ± 0.5% and 98.2

383 ± 0.2%, respectively.

384 Using a simple freezing method, percentages of viable sperm in cryopreserved

385 spermatophores between the control and three-treated groups at 5 min post-plunging in liquid

386 nitrogen were in the ranges of 99.6 ± 0.5% to 99.6 ± 0.7%, similar to those of freshly collected

387 spermatophores (99.7 ± 0.4%; Fig. 4). There were no significant differences (P > 0.05) in

388 percentages of viable sperm in spermatophores among treatments during a 12-month

389 cryostorage. Average percentages of viable sperm in spermatophores in the control and

390 spermatophores treated with either 0.1% PS, moringa or ginger extract at 12-month of
 18

391 cryostorage were 98.3 ± 0.3%, 98.9 ± 0.4%, 98.8 ± 0.5% and 99.3 ± 0.3%, respectively, which

392 were not significantly different (P > 0.05; Fig. 4).

393

394 3.5 Effect of medicinal plant extracts on bacteriological quality

395 3.5.1 Spermatophores cryopreserved using a programmable controlled-rate freezer

396 Number of CHB in freshly collected spermatophores was 3.3 ± 0.6 x 103 CFU g-1 (Fig.

397 5a) while culturable Vibrio was undetectable in this study. At 5 min post-plunging the vials into

398 liquid nitrogen, CHB numbers in spermatophores in the control, 0.1% PS-, moringa extract-, and

399 ginger extract-treated groups were 1.0±0.0, 1.5 ± 0.5, 1.3 ± 0.2 and 2.0 ± 0.0 x 103 CFU g-1,

400 respectively. Numbers of CHB decreased significantly (P < 0.05) during a 12-month

401 cryostorage. Undetectable CHB was observed in spermatophores treated with moringa extract

402 after 12-month cryostorage (Fig. 5a). Number of CHB in cryopreserved spermatophores treated

403 with ginger extract at 12-month cryostorage was 1.0 ± 0.0 x 102 CFU g-1, significantly lower than

404 those of the control (1.5 ± 0.5 x 102 CFU g-1) and 0.1% PS-treated groups (2.0 ± 0.0 x 102 CFU

405 g-1). Freshly collected spermatophores and cryopreserved spermatophores in the control group

406 comprised six bacterial species, e.g. Kocuria palustris, Staphylococcus cohnii subsp. cohnii,

407 Bacillus firmus, B. megaterium, B. pantothenticus and B. pasteurii. At 12-month cryostorage,

408 supplementation of 0.1% PS in the extender resulted in the absence of S. cohnii subsp. cohnii, B.

409 firmus, B. megaterium and B. pantothenticus in post-thawed spermatophores. B. pasteurii was

410 the only bacterial species alive when ginger extract was applied in cryopreservation of

411 spermatophores whereas moringa extract could eliminate all isolated bacterial species

412 contaminated in post-thawed spermatophores.

413 3.5.2 Spermatophores cryopreserved using a simple freezing method

 19

414 Number of CHB in freshly collected spermatophores was 2.0 ± 1.0 x 103 CFU g-1, not

415 significantly different (P > 0.05) compared with those of the control, moringa- and ginger

416 extract-treated groups ranging from 2.0 ± 0.2, 1.5 ± 0.5, 1.5 ± 0.5 x 103 CFU g-1 after 5 min post-

417 plunging the vials into liquid nitrogen (Fig. 5b). A significant reduction in CHB numbers was

418 observed in spermatophores treated with 0.1% PS additive (2.5 ± 1.5 x 102 CFU g-1) after 5 min

419 post-plunging. There was a significant reduction (P < 0.05) in CHB numbers during a 12-month

420 cryostorage of spermatophores in the control and plant-extract-treated groups, except 0.1% PS-

421 treated group. At the12 month post-cryostorage, CHB numbers were not significantly different

422 (P > 0.05 among treatments. Freshly collected spermatophores and cryopreserved

423 spermatophores in the control group consisted of B. firmus, B. insolitus, B. lentimorbus, B.

424 macquariensis, B. thuringiensis, B. pantothenticus, B. pasteurii, B. megaterium, Plesiomonas

425 shigelloides and Acinetobacter lwoffii. At 12-month cryostorage, addition of 0.1% PS resulted in

426 the absence of a few bacterial species, e.g. B. megaterium and B. thuringiensis and A. lwoffii.

427 There were five bacterial species inhibited in cryostored spermatophores treated with ginger

428 extract: B. pantothenticus, B. thuringiensis, B. megaterium, A. lwoffii and P. shigelloides while

429 only P. shigelloides was eliminated in cryostored spermatophores treated with moringa extract

430 supplement. In this study, none of culturable Vibrio was isolated from cryopreserved

431 spermatophores in the control and all treated groups.

432

433 4. Discussion

434 4.1 Effect of cryoprotectants on sperm viability

435 We developed a successful method for cryostorage of F. merguiensis spermatophores.

436 One of the most challenge steps in designing a cryopreservation method is related with
 20

437 optimizing cryoprotectant concentrations and exposure times. Although cryoprotectants prevent

438 cell injury during a freezing-thawing cycle, the lethal effect of each cryoprotectant must be

439 considered. In our study, DMSO (≤ 10%) showed a minimal lethal effect to F. merguiensis

440 sperm. These results were similar to the reports focused on P. monodon sperm by which a

441 relatively low toxicity of DMSO was confirmed in comparison with MeOH, PG, FM and

442 ethylene glycol (Bart et al., 2006; Vuthiphandchai et al., 2007). Due to its low toxicity, DMSO at

443 low concentrations has been used as a protective cryoinjury agent in spermatophore

444 cryopreservation of decapod crustaceans e.g. Ridgeback rock shrimp (Anchordoguy et al., 1988),

445 mud crab, Scylla serrata (Bhavanishankar and Subramoniam, 1997), black tiger shrimp (Bart et

446 al., 2006; Vuthiphandchai et al., 2007) and whiteleg shrimp (Chao et al., 2009). Recently,

447 cryopreservation success of F. merguiensis spermatophores using MgCl2 as a suitable

448 cryoprotectant was reported by Memon et al. (2012), who also pointed out that exposure to

449 DMSO (5-20%) by 24 h exhibited a less toxicity to sperm compared to MeOH, glycerol and

450 ethylene glycol. Although sucrose and MeOH were moderately toxic and other tested

451 cryoprotectants, e.g. PG, FM and Ace were found to be very less effective in protecting sperm

452 from the toxicity due to extremely low sperm viability in the present study, they were also

453 known to protect sperm cells from freezing damages in other aquatic species. For example, in

454 cryopreservation of sperm suspension, spermatic mass and spermatophore of L. vannamei,

455 MeOH was found to be the most effective cryoprotectant to retain sperm survival in comparison

456 with DMSO, glycerol and ethylene glycol (Lazcano et al., 2004). Sucrose has been proven to be

457 less toxic to P. monodon embryos due to maximizing hatching rate when exposure to low

458 concentration (≤ 10%; Vuthiphandchai et al., 2005). Different vulnerability on cryoprotectants of

459 penaeid shrimp among these studies reflects a highly species-dependence.

 21

460 The use of a combination of multiple cryoprotectants results in a decrease in the toxicity

461 to sperm of several penaeid species. As an example, a combination between either DMSO (5%)

462 and glycerol (5%) or DMSO (5%) and trehalose (5%) was effective in protecting against

463 cryoinjury owing to retaining the highest viability (61-87%) of post-thawed sperm of Indian

464 white shrimp (Diwan and Joseph, 1999). In M. rosenbergii spermatophore cryopreservation, an

465 incorporation of DMSO (10%) and PG (10%) generated higher viability, compared to those of

466 DMSO, PG, MeOH, glycerol and ethylene glycol when treated alone (Valentica claudet et al.,

467 2016). In contrast, none of sperm viability was improved when an addition of 5% DMSO

468 combined with sucrose, proline or glycerol into extender was performed during cryopreservation

469 of Ridgeback rock shrimp (Anchordoguy et al., 1988). Similar results were observed in our study

470 supported by a significant reduction in post-thawed sperm viability of spermatophores suspended

471 in 5% sucrose solution combined with 5% DMSO in comparison with 5% DMSO alone. In

472 addition, administration pattern of mixed cryoprotectants was interestingly found to influence

473 post-thawed sperm viability due to a significantly higher sperm viability obtained in this study

474 when simultaneous addition of cryoprotectants was conducted. In spite of protective mechanisms

475 still unknown at this stage, interactions of mixed cryoprotectants (simultaneous and non-

476 simultaneous additions) and sperm cells may account for such a phenomenon and merit to be

477 further investigated. In the present study, regardless of concentrations and types of

478 cryoprotectants, a pronounced effect on the loss of sperm viability was obviously observed when

479 exposure time increased. This suggested that equilibration time is of paramount importance to

480 allow penetration of cryoprotectants into the biological materials. The use of DMSO at low

481 concentrations or shorter equilibration time in F. merguiensis sperm in the present study, like 5%

482 DMSO for 20 min or 10% DMSO for 10 min, would reduce sperm mortality rate. In P.
 22

483 monodon, substantial declines in sperm viability were observed when spermatophores were

484 exposed to stepwise equilibrium times (10, 20, 30 and 60 min) and increased concentrations (5,

485 10, 15 and 20%) of different cryoprotectants (DMSO, PG, FM, MeOH and ethylene glycol;

486 Vuthiphandchai et al., 2007). Gwo and Lin (1998) reported that lower survival rate of Penaeus

487 japonicus embryos was a consequence of prolonging equilibrium times from 10 to 30 min in all

488 cryoprotectants examined at 10% concentration.

489

490 4.2 Optimal freezing rate

491 Freezing rate is a critical factor in sperm cryopreservation of F. merguiensis as well as

492 concentration and exposure time of cryoprotectants. Sperm cells of F. merguiensis were

493 extremely vulnerable to fast freezing rate regardless of cryoprotectants, evidenced by a

494 significant reduction in sperm viability at higher freezing rates applied. In general, fast freezing

495 protocol creates lethal cryodamages to sperm cells by alteration of actin polymerization within

496 the cytoskeleton leading to more susceptibility to osmotic change (Herraez et al., 2012) and by

497 cellular membrane rupture resulting from intracellular ice formation (Woods et al., 2004). On the

498 contrary, slow freezing rate generates variable survivals dependent on cryoprotectant and

499 biological material used because it forms ice crystals in the extracellular regions (Woods et al.,

500 2004). In fact, slow freezing protocols have been used successfully in spermatophore

501 cryopreservation of decapod species such as sperm suspension of Ridgeback rock shrimp

502 (Anchordoguy et al., 1988), Indian white shrimp (Diwan and Joseph, 1999), whiteleg shrimp

503 (Lezcano et al., 2004), or spermatophores of black tiger shrimp (Vuthiphandchai et al., 2007) and

504 whiteleg shrimp (Chao et al., 2009). Vuthiphandchai et al. (2007) confirmed a role of freezing

505 rate in cryopreservation by which P. monodon spermatophores were frozen with faster (-4 to -16
 23

506 °C min-1) and slower (<-4 °C min-1) freezing rates from 25 °C to a final temperature of -80 °C

507 with 5% DMSO additive. They observed the incidence of high sperm survival when

508 spermatophores were frozen at -2 °C min-1 while complete mortality of sperm was recorded

509 when faster freezing rates were used. A slow freezing rate at -1.5 °C min-1 with the mixture of

510 10% DMSO and 10% PG seemed to be amenable for spermatophore cryopreservation of M.

511 rosenbergii due to retaining higher viable sperm and fertilization potential than at -3 and -5 °C

512 min-1 (Valentica claudet et al., 2016). In accord, Selvakumar et al. (2018) also revealed the

513 effects of freezing rate on cryoinjuries of F. indicus spermatophores frozen with various freezing

514 rates of -0.5 °C min-1, -10 °C min-1 and vitrification and found a significant increase in

515 percentages of cell membrane integrity and DNA integrity of sperm corresponding with a slow

516 freezing rate at -0.5 °C min-1 employed.

517

518 4.3 Optimal thawing temperature and duration

519 Thawing protocol is a key factor in cryopreservation success depending usually on the

520 biological materials and volume of frozen samples (Herraez et al., 2012). The thawing

521 temperatures and durations affect sperm survivability. Thawing of cryostored spermatophores of

522 F. merguiensis in a water bath at 30 °C for 2 min was recommended in this study. Similar result

523 was confirmed by Memon et al. (2012) who suggested that a slow thawing protocol at 27 °C for

524 2 min was suitable for F. merguiensis spermatophores stored in liquid nitrogen. Frozen

525 spermatophores of penaeid shrimp were usually thawed in a 30-35 °C water bath for 1-2 min

526 (Chow et al., 1985; Vuthiphandchai et al., 2007; Chao et al., 2009; Valentica claudet et al., 2016;

527 Selvakumar et al., 2018). In the present study, a decrease in sperm survival was directly related

528 with increased thawing temperatures, which may have been liked to loss of cell membrane
 24

529 integrity, decreased mitochondrial activities, cytoskeleton damages and DNA fragmentation

530 induced by a freezing-thawing cycle (Herraez et al., 2012).

531

532 4.4 Long-term storage and effect of medicinal plant extracts on sperm survival

533 The presence of relatively high post-thawed sperm viability of cryopreserved

534 spermatophores of F. merguiensis during a 12-month cryostorage indicated an establishment of

535 good quality of cryopreserved spermatophores in the sperm bank. In accordance with

536 Akarasanon et al. (2004), M. rosenbergii spermatophores cryopreserved up to 200 d exhibited

537 high sperm survival rate even though their fertilizing abilities significantly declined when being

538 stored more than 150 d. Vuthiphandchai et al. (2007) reported high post-thawed sperm viability

539 (87%) and fertilization rate (72%) of P. monodon spermatophores cryostored for 60-62 d which

540 were not significantly different from those of freshly collected spermatophores. However, they

541 claimed that P. monodon spermatophores could be preserved up to 210 d in liquid nitrogen tank

542 in spite of low sperm viability (< 40%). Recently, Memon et al. (2012) observed that F.

543 merguiensis spermatophores kept in liquid nitrogen tank for ≤ 90 d maintained moderate sperm

544 viability (> 55%) and fertilizing ability (64%) while during a 90-180 d cryostorage, sperm

545 viability declined significantly.

546 We have developed a simple, affordable and effective cryopreservation protocol for F.

547 merguiensis spermatophores based on freezing at 4-cm above liquid nitrogen surface

548 (corresponding to -2.5 °C min-1). A few reports have confirmed cryopreservation success of

549 decapods spermatophores using a practical method. One such species is M. rosenbergii whose

550 spermatophores were cryopreserved with 20% ethylene glycol at -1.5 to -2.5 °C min-1 of freezing

551 rates using dry-ice cubes in the 95% ethanol and nitrogen vapor, which could retain high sperm
 25

552 survival and moderate levels of fertilization capacity up to 150-d storage (Akarasanon et al.,

553 2004). Likewise, Chow et al. (1985) suspended M. rosenbergii spermatophores in sperm

554 extender containing 10% glycerol before pre-cooling with liquid nitrogen vapor for 5-10 min,

555 and observed fertilized eggs when artificial insemination was performed after cryostorage in

556 liquid nitrogen for 31 d. However, Lazcano et al. (2004) reported a failure in cryopreservation of

557 L. vannamei spermatophore using nitrogen vapor at a cooling rate of approximately -10 °C min-1

558 due to reversion of spermatic cells despite cryoprotectant utilized. Other studies confirmed a

559 successful cryopreservation protocol by cooling at -2 °C min-1 from 25 °C to -80 °C with 5%

560 DMSO additive for P. monodon spermatophores (Vuthiphandchai et al., 2007), at -1.5 °C min-1

561 with mixture of 10% DMSO and 10% PG for M. rosenbergii spermatophores (Valentica claudet

562 et al., 2016) and at -0.5 °C min-1 with 5% DMSO plus 5% methanol for F. indicus

563 spermatophores (Selvakumar et al., 2018).

564 Our study confirmed that moringa and ginger extracts were harmless to viable F.

565 merguiensis sperm cryopreserved in liquid nitrogen, evidenced by no significant differences in

566 percentages of viable sperm detected between the control and plant-extract supplemented groups.

567 As far as we can ascertain, this is, for the first time, a report focused on plant extract application

568 in spermatophore cryopreservation of penaied shrimp to maintain high sperm quality and

569 eliminate bacterial contaminants. Mechanism interaction between plant extract and shrimp sperm

570 is unknown. Several studies pointed out that administration of moringa and ginger

571 extracts/powders provided beneficial effects on reproductive systems of Wistar rat (Rattus

572 norvegicus) by improving sperm concentration, motility, viability and plasma testosterone

573 (Khaki et al., 2009; Akunna et al., 2012). They postulated that strong antioxidant substances in

574 the extracts mitigate or prevent the formation of free radicals. In the perspective of reproductive
 26

575 system improvement in aquatic animals, dried ground leaves of moringa aided to enhance

576 growth, fecundity and embryo viability in zebrafish, Danio rerio (Paul et al., 2013). Although

577 high sperm survival of F. merguiensis in the present study was observed during a 12-month

578 cryostorage, further study is required to elucidate fertilization capacity of cryopreserved F.

579 merguiensis spermatophores supplemented with medicinal plant extracts to ensure fertilization

580 success and guarantee the use of cryopreservation protocol of F. merguiensis spermatophores. As

581 a subcellular injury of the sperm cells may result in the reduction of fertilization capacity, further

582 study associated with structural architecture at microscopic level, biochemical changes upon

583 cryopreservation are also required for freezing of F. merguiensis spermatophores for the

584 breeding program.

585

586 4.5 Effect of medicinal plant extracts on bacteriological quality in cryopreserved sperm

587 Freshly collected spermatophores of marine shrimp was reported to have bacterial flora

588 number of 46.3 x 104 CFU g-1 (Nimrat et al., 2008). In the present study, an appearance of high

589 CHB numbers in cryostored spermatophores by 3 months of cryostorage, and thereafter followed

590 by a significant decrease may reflect a chilling sensitivity of contaminated bacteria (Gram and

591 Dalgaard, 2002). Nimrat et al. (2008) observed high CHB number of ca. 105 CFU g-1 in 1-h

592 cryostored spermatophores of P. monodon spermatophores and an apparent decline in CHB

593 number (95% reduction) after 30-d storage. Several bacterial species isolated from freshly

594 collected spermatophores in the present study were generally ubiquitous bacteria, e.g. K.

595 palustris, S. cohnii subsp. cohnii, B. firmus, B. megaterium, B. pantothenticus, B. pasteurii. B.

596 firmus, B. insolitus, B. lentimorbus, B. macquariensis, B. thuringiensis, B. pantothenticus, B.

597 pasteurii, P. shigelloides and A. lwoffii. Similarly, a considerable bacterial genera was frequently
 27

598 found in penaied spermatophores including Staphylococcus, Bacillus, Pseudomonas aeruginosa,

599 Micrococcus, Paracoccus, Ruegeria, Proteus, Enterobacter, Vibrio and non-fermentative Gram

600 negative bacilli (Nimrat et al., 2005, 2008; Ueno et al., 2015). In the present study, moringa and

601 ginger extracts could eliminate several species of bacterial flora isolated from cryostored of

602 banana shrimp. The antibacterial effects of the extracts are expected perhaps because of

603 phytochemical constitutes which have been proven to have broad-spectrum antibacterial

604 potentials. The major antimicrobial components in ginger extract are gingerol, shogaol,

605 zingerone and paradol (Rahmani et al., 2014) while ethanolic extract from moringa leaves has

606 been claimed to contain alkaloids, polyphenols, flavonoids, anthraquinones, coumarins, tannins,

607 triterpenes, saponins, antimicrobial peptides and some secondary metabolites as bioactive

608 antibacterial components (Wang et al., 2016). Interestingly, moringa and ginger extracts had

609 inhibitory activity against potential human pathogens contaminated in spermatophores in the

610 present study. P. shigelloides is pathogenic in humans and considered as a causative agent of

611 gastroenteritis due to consuming raw/undercooked seafood or exposure to coastal waters

612 (Kahraman et al., 2017). A. lwoffii, ubiquitous in the environments, is causative agent for

613 nosocomial infection especially in immunocompromised patients (Mittal et al., 2015).

614 Despite, no bacterial effect on shrimp sperm quality studied, Alfaro et al. (1993) reported

615 pathogen infection, e.g. Vibrio alginolyticus and Pseudomonas putrefaciens, is associated with

616 melanized formation of the male reproductive tissues of captive Litopenaeus setiferus and male

617 infertility, which consequently resulted in a decrease in seed production. In cryostored sperm of

618 fish, fish pathogens, Aeromonas hydrophila and P. fluorescens, caused a degenerative change in

619 sperm motility and viability, fertilization capacity and sperm morphometry, and enabled a

620 vertical transmission to embryos through artificial insemination of cryopreserved sperm of silver
 28

621 barb, Barbodes gonionotus (Boonthai et al., 2016b; 2018). Therefore, the effects of bacterial

622 infection on shrimp sperm quality should be further investigated.

623

624 5. Conclusions

625 An optimized cryopreservation protocol using a programmable controlled-rate freezer

626 would help sperm bank authorities to manage male gamete of F. merguiensis for aquaculture,

627 create preservation strategy of desirable breeding traits, and facilitate hatchery management e.g.

628 domestic and international transports of good-quality spermatophores. A long-term cryostorage

629 of F. merguiensis spermatophores with moringa or ginger extract supplements (0.1 mg mL-1

630 each) up to 12 months could satisfactorily retain high sperm survivability and reduce the

631 abundance of bacterial contaminants. Cryopreservation technique using a non-programmable

632 protocol of F. merguiensis spermatophores is practical and cost-effective to perform in

633 commercial hatcheries and would facilitate hatchery production of F. merguiensis seed to meet

634 the market need. The interest of this study lies in the fact that cryopreservation protocol of F.

635 merguiensis sperm with moringa or ginger extract supplements for long-term storage would be a

636 valuable tool for hatchery operations to maintain a continuous and reliable supply of male

637 gamete stocks without biosafety concern.

638

639 Funding

640 This work was supported by the National Research Council of Thailand (grant no.

641 4/2556, 3/2560, 40/2561). We would like to thank the staffs of Department of Microbiology and

642 Department of Aquatic Science, Faculty of Science, Burapha University for technical support.

643
 29

644 Ethics approval

645 All protocols used in this study involving the use of live shrimp were approved for

646 animal ethics by the Burapha University Animal Care and Use Committee.

647

648 Declarations of interest

649 The authors declare that we have no conflict of interests.

650

651 Author contributions

652 S. Nimrat wrote the research proposal, set up the experimental design, supervised the

653 project, analyzed and interpreted data, and edited and revised the manuscript as well as

654 performing bacteriological study of spermatophores during cryostorage. S. Noppakun performed

655 the experiment about the cryoprotectant toxicity. K. Sripuak conducted the experiment on

656 development of the freezing protocols. T. Boonthai performed interpretation of results and

657 statistical analysis and assisted in drafting the manuscript. V. Vuthiphandchai conducted the

658 experiments about cryopreservation protocol with supplementation of medicinal plant extracts

659 for long-term storage of spermatophores using a programmable controlled-rate freezer and a

660 simple freezing method as well as collected shrimp and spermatophores and drafted the

661 manuscript.

662

663 References

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801
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802 Table legend

803 Table 1 Percentage of sperm viability of Fenneropenaeus merguiensis spermatophores

804 cryostored with different cryoprotectants and cooling rates from 25 °C to -80 °C.

805 Table 2 Percentage of sperm viability of cryopreserved Fenneropenaeus merguiensis

806 spermatophores thawed at different rates.

807

808 Figure caption

809 Fig. 1 Sperm viability (%) of Fenneropenaeus merguiensis spermatophores equilibrated with

810 different cryoprotectants, concentrations, and exposure periods. Bars with superscript

811 letters in the same treatment indicate significant difference (P < 0.05) over the time. Bars

812 with superscript numbers within each exposure period indicate significant difference (P <

813 0.05) among treatments. Sperm viability of freshly collected spermatophores suspended in

814 Ca-F saline without cryoprotectant was used as the control to ensure the spermatophores

815 having good quality prior to being exposed to each cryoprotectant. Sperm viabilities in the

816 control group in the ranges of 98.6 ± 2.1 to 99.8 ± 1.3% were recorded before the

817 experiment started. DMSO: dimethyl sulfoxide, MeOH: methanol, PG: 1,2-propylene

818 glycol, FM: formamide, Ace: acetamide.

819 Fig. 2 Post-thawed sperm cells of Fenneropenaeus merguiensis showing intact membrane

820 structure: unstained alive sperm (arrow) and shrinking sperm with pink-purple staining

821 (arrowhead), with inset showing cell membrane rupture of sperm due to fast thawing rate

822 protocol.
 37

823 Fig. 3 Sperm viability (%) of Fenneropenaeus merguiensis spermatophores cryopreserved with

824 either penicillin-streptomycin or plant extract additives during a 12-month cryostorage

825 using a programmable controlled rate freezer.

826 There were no significant differences (P < 0.05) observed among treatments within the

827 same sampling period and over time.

828 Fig. 4 Sperm viability (%) of Fenneropenaeus merguiensis spermatophores cryopreserved with

829 either penicillin-streptomycin or plant extract additives during a 12-month cryostage using

830 a simple practical method.

831 There were no significant differences (P < 0.05) observed among treatments within the

832 same sampling period and over time.

833 Fig. 5 Numbers of culturable heterotrophic bacteria (CFU g-1) in Fenneropenaeus merguiensis

834 spermatophores cryopreserved with either penicillin-streptomycin or plant extract

835 additives during a 12-month cryostorage using (a) a programmable controlled rate freezer

836 and (b) a simple freezing method.

837 Bars with superscript letters within each sampling time indicate significant difference (P <

838 0.05) among treatments. Bars with superscript numbers within the same treatment indicate

839 significant difference (P < 0.05) over time.

Table 1 Percentage of sperm viability of Fenneropenaeus merguiensis spermatophores cryostored with different cryoprotectants and cooling

rates from 25 °C to -80 °C

5 min post-cryostorage 1 h post-cryostorage

Simultaneous
Cooling rates Simultaneous addition Addition of 5%
addition of 5%
(°C min-1) 5% DMSO 5% Sucrose of 5% sucrose and 5% sucrose, and then 5% DMSO
sucrose and 5%
DMSO 5% DMSO
DMSO

-1 °C min-1 63.1 ± 1.9 b,2 50.4 ± 2.6 c,2 86.2 ± 7.7 a,1 68.0 ± 0.9 b,1 73.4 ± 2.1 a,2 67.7 ± 3.9 b,1

-2 °C min-1 89.9 ± 2.5 a,1 79.6 ± 3.6 b,1 82.9 ± 3.7 a,1 47.1 ± 3.5 c,3 83.5 ± 2.7 a,1 61.4 ± 1.2 b,1

-3 °C min-1 26.2 ± 4.0 b,3 3.5 ± 0.4 c,4 65.3 ± 10.4 a,2 55.9 ± 0.7 a,2 23.3 ± 0.6 b,3 40.6 ± 2.7 a,2

-4 °C min-1 2.7 ± 1.0 c,4 38.4 ± 5.9 b,3 1.7 ± 0.1 c,3 57.9 ± 8.8 a,2 7.2 ± 1.1 a,4 2.2 ± 0.3 a,3

Data were expressed as mean ± S.D. Means with superscript letters in the same row and storage period indicate significant difference (P < 0.05)

among cryoprotectants tested. Means with superscript numbers in the same column indicate significant difference (P < 0.05) among cooling

rates.
Table 2 Percentage of sperm viability of cryopreserved Fenneropenaeus merguiensis

spermatophores thawed at different rates

Thawing temperature and duration Sperm viability (%)

Freshly collected spermatophores 88.4 ± 0.8 a

30 °C for 2 min 85.1 ± 1.4 a

50 °C for 2 min 61.6 ± 0.8 b

70 °C for 2 min 25.3 ± 1.2 c

90 °C for 2 min 15.8 ± 1.8 d

Data were expressed as mean ± S.D. Means with superscript letters in the column indicate

significant difference (P < 0.05) among thawing rates.

Highlights of the manuscript

- This study reports, for the first time, the cryopreservation protocol using a

programmable controlled-rate freezer for banana shrimp (Fenneropenaeus merguiensis)

spermatophores.

- This study also develops a practical method for cryopreservation of banana shrimp

spermatophores using a Styrofoam box, which would provide the simpler and more affordable

cryopreservation protocol for hatchery operations.

- This study, for the first time, reports the potential use of extracts from moringa

(Moringa oleifera Lam.) and ginger (Zingiber officinale Roscoe) as alternative antimicrobial

agents for controlling bacterial contaminants in cryopreserved spermatophores of penaeid shrimp

to reduce the use of harmful pharmaceuticals in aquaculture and provide a more reliable supply

of spermatophores without a biosafety concern.

Declarations of interest

The authors declare that we have no conflict of interests.