Mouse sperm delivery via cauda epididymis without ... · Mouse sperm delivery via cauda epididymis without transport medium Man-Xi Jiang Corresp ., 21, 1Mei-Shan Wang 2 , Corresp.Xiang-Hong

Mouse sperm delivery via cauda epididymis without transportmediumMan-Xi Jiang Corresp., 1 , Mei-Shan Wang 2 , Xiang-Hong Ou 1 , Xue-Jin Chen 2 , Yan Zhu Corresp. 3

1 Human Reproductive Centre, Guangdong No.2 Provincial People’s Hospital, Guangzhou, Guangdong Province, China2 Department of Laboratory Animal Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China3 Medical Research Centre, Guangdong No.2 Provincial People's Hospital, Guangzhou, China

Corresponding Authors: Man-Xi Jiang, Yan ZhuEmail address: [email protected], [email protected]

This study was aimed to investigate the effects of room temperature (RT, 20-25 oC) andabsence of medium during cauda epididymis transport on spermatozoa quality, fertilityand embryo development. In the first experiment, fresh sperm from one side of caudaepididymis was used for in vitro fertilization, and another side was delivered at RT or 4-8 oCeither with or without M2. In the second experiment, each side of cauda epididymisobtained from the same mouse was individually delivered at RT or 4 oC with or without M2.Finally, sperm motility, progressive motility scores and fertility of fresh spermatozoa orthose from transported cauda epididymis, and IVF embryo development were evaluated.Progressive motility scores and fertilization rates were higher in fresh spermatozoa thantransported sperm; sperm motility of transported cauda epididymis at 4-8 oC wascomparable to fresh spermatozoa, but spermatozoa motility of transported caudaepididymis at RT was inferior to fresh spermatozoa. Spermatozoa motillty of transportedcauda epididymis at 4-8 oC with transport medium was much higher than that withouttransport medium; absence of transport medium did not affect sperm motility oftransported cauda epididymis at 4-8 oC but affected sperm motility of transported caudaepididymis at RT. Sperm quality from transported cauda epididymis can be efficiently keptat 4-8 oC, and cauda epididymis transport at 4-8 oC without M2 is more beneficial onkeeping their fertility. Moreover cauda epididymis transport at RT without medium couldsufficiently produce embryos for obtainning live offsprings.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2542v1 | CC BY 4.0 Open Access | rec: 21 Oct 2016, publ: 21 Oct 2016

1 Title: Mouse Sperm Delivery via Cauda Epididymis without Transport Medium Running head:

2 Transport of Mouse Cauda Epididymis

3 Authors：Man-Xi Jiang1*, Mei-Shan Wang2, Xiang-Hong Ou1, Xue-Jin Chen2 and Yan Zhu1*

4 1 Guangdong No.2 Provincial People’s Hospital, Guangzhou 510317, China

5 2 Department of Laboratory Animal Sciences, Shanghai Jiao Tong University School of

6 Medicine, Shanghai 200025, China

7 *Corresponding authors: Man-Xi Jiang and Yan Zhu

8 Email: [email protected] and [email protected]

9 Tel: +86 20 89168746

10 Address: No.466 of Xingang Zhong Rd, Haizhu District, Guangzhou 510317, China

11 Abstract

12 This study was aimed to investigate the effects of room temperature (RT, 20-25 oC) and absence

13 of medium during cauda epididymis transport on spermatozoa quality, fertility and embryo

14 development. In the first experiment, fresh sperm from one side of cauda epididymis was used

15 for in vitro fertilization, and another side was delivered at RT or 4-8 oC either with or without

16 M2. In the second experiment, each side of cauda epididymis obtained from the same mouse was

17 individually delivered at RT or 4 oC with or without M2. Finally, sperm motility, progressive

18 motility scores and fertility of fresh spermatozoa or those from transported cauda epididymis,

19 and IVF embryo development were evaluated. Progressive motility scores and fertilization rates

20 were higher in fresh spermatozoa than transported sperm; sperm motility of transported cauda

21 epididymis at 4-8 oC was comparable to fresh spermatozoa, but spermatozoa motility of

22 transported cauda epididymis at RT was inferior to fresh spermatozoa. Spermatozoa motillty of

23 transported cauda epididymis at 4-8 oC with transport medium was much higher than that without


mailto:[email protected]

mailto:[email protected]

24 transport medium; absence of transport medium did not affect sperm motility of transported

25 cauda epididymis at 4-8 oC but affected sperm motility of transported cauda epididymis at RT.

26 Sperm quality from transported cauda epididymis can be efficiently kept at 4-8 oC, and cauda

27 epididymis transport at 4-8 oC without M2 is more beneficial on keeping their fertility. Moreover

28 cauda epididymis transport at RT without medium could sufficiently produce embryos for

29 obtainning live offsprings.

30 Key words: Cauda epididymis; Transport medium; Sperm motility; Fertility; Embryonic

31 development


33 Introduction

34 Although both cryopreserved embryos and sperm can be used to deliver or distribute animals,

35 cryopreserved samples require a special container for delivery in order to maintain them at the

36 requisite low temperatures. However, the cryopreserved sperm with liquid nitrogen requires

37 bacteriological monitoring before transport registration and several procedures are necessary to

38 obtain pups (i.e. thawing of frozen spermatozoa and subsequent IVF using frozen–thawed

39 spermatozoa), which are often laborious and time consuming.

40 Fresh sperm delivery is ideal and does not require special containers such as dry shippers;

41 however, sperm can be stored at room or refrigerated temperature for only a short duration

42 (Fuller & Whittingham 1996; Sato & Ishikawa 2004; Sato et al. 2001). Several recent reports

43 have described the successful transport of mouse sperm as fresh epididymal sperm at refrigerated

44 temperatures (Jishage et al. 1997; Kaneko et al. 2009b; Mochida et al. 2005; Sankai et al. 2001;

45 Sato & Ishikawa 2004; Sato et al. 2001; Takeo et al. 2012), and transporting epididymal sperm is

46 considered to be an easier method that could circumvent the problems associated with the

47 delivery of cryopreserved embryos and sperm.

48 Recently, the potential for storage of gametes and gonadal tissues under non-freezing conditions

49 has been recognized in several species (An et al. 1999; Sankai et al. 2001; Sato & Ishikawa

50 2004). The method does not require any specialized freezing devices or technical skills. Mouse

51 spermatozoa that have been retrieved postmortem and stored at low temperature for a few days

52 can subsequently be used to fertilize oocytes, and the resultant embryos develop normally (An et

53 al. 1999; Sankai et al. 2001). Even in humans, posthumous sperm retrieval (PSR) is an

54 unconventional technology (Batzer et al. 2003; Rothman 1980); since then, only a few

55 pregnancies and births attributed to PSR-derived sperm have been reported (Batzer et al. 2003;


56 Belker et al. 2001; Check et al. 1999).

57 In addition, a previous study specifically evaluated the effect of several media on sperm motility

58 and found M2 was the most suitable media for sperm storage at 22 oC (Sato & Ishikawa 2004).

59 Takeo et al. (Takeo et al. 2012)compared the effects of other media including M2 on sperm

60 fertility and demonstrated Lifor was superior to M2 medium for maintaining the fertility of cold

61 storaged sperm. Therefore researches have so far proved inconclusive about which medium is the

62 best one for maintaining sperm motility and fertility during storage or transport.

63 In the present study, we transported cauda epididymis at room temperature (RT, 20-25 oC) or at

64 4-8 oC with or without transport medium M2 to maintain humidity, compared the influences of

65 transport medium-free and room tempreature on motility, progressive motility scores and fertility

66 of spermatozoa from the transported cauda epididymis, and the development of resulting embryo

67 was evaluated.

68 Materials and methods

69 Humane care and use of animals

70 We obtained 6- to 8-week-old ICR mice and 6-week-old female BDF1 mice from Guanzhou

71 Animals Center (Guangzhou, China) and bred at the Guangdong No.2 Provincial People’s

72 Hospital (Guangzhou, China). The animal procedures were approved by Guangdong No.2

73 Provincial People’s Hospital, and this study was carried out in strict accordance with the Ethical

74 Committee of the Guangdong NO.2 Provincial People’s Hospital (No. 2014-KYLLM-065). For

75 cauda epididymis delivery at different temperature and transport medium conditions, 10-week-

76 old male BDF1 mice were used. The cauda epididymides of the BDF1 mice were collected in

77 our collaborators’ laboratories, the Department of Laboratory Animal Science, Shanghai Jiao

78 Tong University School of Medicine, which is over 2,000 km away from our facility. All the


79 cauda epididymides were carried to the Guangdong No.2 Provincial People’s Hospital by train or

80 long-distance coach strictly within 24 hours after the samples were collected by a designated

81 techincian.

82 Reagents

83 Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were

84 purchased from Ningbo Sansheng Hormone Factory (Ningbo, China). All other chemicals were

85 obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO), unless otherwise stated.

86 Experimental design

87 Two experiments were performed in the present study. In the first experiment (as shown in

88 Figure 1), cauda epididymal samples were divided into four groups (Groups 1–4) and each group

89 was subjected to a different treatment. In each group, all fresh sperm was obtained from one side

90 (A) of the cauda epididymis for IVF, while the another side (B) was transported either with or

91 without medium, depending on the treatment group development between fresh sperm and

92 transported cauda epididymis from the same mouse. In group 1- 4, side B of cauda epididymis

93 were transported at RT without M2, at 4–8°C without M2, in M2 at RT and in M2 at 4-8 °C,

94 respectively. We finally compared the differences of sperm motility, progressive motility scores,

95 fertilization rates and IVF embryo development. In the second experiment (as shown in Figure

96 2), cauda epididymis were divided into four treatment groups (I - IV): In group I, side A and B of

97 cauda epididymis from the same mouse were transported at RT with and without M2,

98 respectively; in group II, both sides (A and B) were transported at 4–8°C with and without M2,

99 respectively; in group III, both sides (A and B) were transported with M2 at 4-8°C and RT,

100 respectively; in group IV, both sides (A and B) were transported without M2 at 4-8°C and RT,

101 respectively. We finally studied the effects of transport temperature and medium of cauda


102 epididymis on sperm motility, progressive motility scores, fertilization rates, and the

103 development of IVF embryos.

104 Cauda epididymis collection and transport

105 Male BDF1 mice were euthanized by cervical dislocation at collaborator’s laboratory. A small

106 incision was made in the scrotum, and the cauda epididymides were collected at 37oC in M2

107 medium. Cauda epididymis for transport were ligated at both ends immediately, apart from the

108 samples for fresh sperm in vitro fertilization (IVF).

109 Every samples for transport were placed in 0.2-mL sterilized tubes either with or without 20 µL

110 M2 as shown in experiment design (Figure 1 and 2). All performances including the following

111 fresh sperm IVF were carried out by an experienced technician from Guangdong No.2 Provincial

112 People’s Hospital. The experiments in each group were repeated at least three times, and all

113 samples must be in accordance with the acceptance criteria.

114 For transport at 4-8 oC, the transport tubes with cauda epididymis were placed into a cardboard

115 box with a temperature tracker that functions as a digital thermometer and data logger (MH-TO-

116 1; Hangzhou Bright Technology Inc., Hangzhou, China) and a sheet of cotton wool (Takeo et al.

117 2012). The box was placed in a vacuum bottle (JMK-500-BK, Thermos Co., USA) containing

118 two gel packs that had been precooled in a refrigerator at 4 oC. The bottle was placed inside a

119 Styrofoam box and covered with four precooled gel packs. If the digital shown on temperature

120 tracker wasn’t between 4 and 8oC, then the transported cauda epididymis sample was directly

121 abandoned.

122 For transport at RT (20-25 oC), the transport tubes containing the cauda epididymis sample were

123 placed in a cardboard box with a temperature tracker, in a vacuum bottle with gel packs

124 prewarmed at 20 oC, and the vacuum thermos flask was placed inside a Styrofoam box and


125 covered with prewarmed gel packs. If the digital shown on temperature tracker wasn’t between

126 20 and 25oC, then the transported cauda epididymis sample was not used for the further

127 experiment.

128 Isolation and capacitation of epididymal spermatozoa

129 Once cauda epididymis arrived at our laboratory within 24 h after collection and met the

130 acceptance criteria, the samples were washed in prewarmed M2 medium at 37 oC at least three

131 times and the spermatozoa were released from the epididymides into a 4-well dish containing

132 400 µL of human tubal fluid (HTF) medium rewarmed at 37 oC immediately. The spermatozoa

133 were capacitated at 37 oC for 30 min in a 5% CO2 atmosphere for assessing sperm motility and

134 progressive motility scores, and in vitro fertilization.

135 Assessment of sperm motility and progressive motility scores

136 The capacitated sperm was diluted tenfold with HTF medium (Millipore) and assessed in a

137 Makler chamber. Sperm motility was determined by microscopic analysis according to the

138 criteria described by Jequier and Crich (Jequier & Crich 1986), and the percentage of actively

139 motile spermatozoa and progressive motility scores were assessed using this method (3 replicates

140 per sample; 100 spermatozoa observed in 5–6 fields per replicate). The motility of sperm was

141 scored on a scale of 0 to 4, where 0 indicates no motility; 1, weak or sluggish motility; 2, definite

142 motility; 3, good motility; and 4, vigorous motility. Samples with scores of 2–4 were then used

143 to calculate the percentage of actively motile spermatozoa.

144 IVF to assess the fertilization ability of spermatozoa

145 Ovulated oocytes were obtained from superovulated C57BL/6 mice (4–6 weeks of age) after

146 intraperitoneal (ip) injection with 7.5 IU PMSG followed by 7.5 IU of hCG at 46–48 h after the

147 first injection. The oocytes were isolated in HTF medium and washed in the same medium three


148 times. A few minutes before fertilization, the oocytes were placed in 500 µL prewarmed HTF

149 and covered with sterile mineral oil.

150 Fresh or transported spermatozoa from cauda epididymis were capacitated as described above

151 were used for IVF (Pease et al. 2011). In brief, the concentration of the capacitated sperm was

152 adjusted to approximately 1–2 × 107 sperm/mL, and 10 µL of the suspension was added to IVF

153 drops containing oocytes to yield a final concentration of about 2 × 105 sperm/mL. This was then

154 incubated at 37 oC for 5–6 h in a humidified atmosphere containing 5% CO2. The presumptive

155 zygotes were washed and cultured in HTF under the same conditions.

156 Two-cell embryos were counted 24 h after insemination. The embryos were then washed in

157 potassium simplex optimized medium with amino acids (KSOM-aa, Millipore), and 10–15

158 embryos were cultured in a drop of the same medium (20 μL) covered with a layer of sterile

159 mineral oil. Embryos that developed into blastocysts were counted at 72 h post-insemination.

160 The percentages of two-cell embryos and blastocysts were calculated using the total number of

161 presumptive zygotes in culture.

162 Embryo transfer

163 All the blastocysts were bilaterlly transferred to the uteri of day 2.5 pseudopregnant ICR mice

164 that had been previously mated with vasectomized ICR male mice. Average 12-18 blastocysts

165 were transferred for each pseudopregnant mouse. Offspring were born naturally or delivered by

166 Cesarean section at day 18.5 of gestation.

167 Statistical analysis

168 Data were analysed using Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS 17

169 (SPSS Inc., Chicago, IL, USA). Fertilization rates of spermatozoa, rates of two-cell embryos

170 developed to blastocyst in vitro and birth rates of offsprings were analyzed by the χ2 test.


171 Spermatozoa motiity and progressive motility scores were analyzed by one-way analysis of

172 variance (ANOVA), respectively. Values were considered statistically different at P values of

173 less than 0.05.

174 Results

175 Spermatozoa motility and progressive motility scores of fresh and transported sperm in cauda

176 epididymis

177 In order to compare the effect of different transport conditions on spermatozoa, we incubated

178 fresh and transported sperm from cauda epididymis in HTF medium at 37 oC for 30 min, and

179 then measured the spermatozoa motility and progressive motility scores .

180 These findings as shown in Figure 3 and Table S1 and S2 demonstrated that the spermatozoa

181 motility significantly differed between fresh and transported cauda epididymal samples, and all

182 of the fresh spermatozoa had considerably higher motility than those transported in cauda

183 epididymis(p<0.05). These results demonstrated that the spermatozoa progressive motility scores

184 of fresh and transported spermatozoa in cauda epididymides at 4-8 oC were comparable in

185 Groups 1 - 4, there are no significant differences (p>0.05), but the progressive motility scores of

186 spermatozoa transported from cauda epididymis at RT was inferior to that of fresh spermatozoa,

187 regardless of the presence or absence of transport medium(p<0.05), suggesting that the higher

188 temperature seems have a more impact on spermatozoa progressive motility scores comparing

189 with the absence of transport medium.

190 Spermatozoa motility and progressive motility scores of transported sperm in cauda epididymis

191 In order to evaluate the effect of transport medium and temperature on cauda epididymis

192 delivery, we compared the sperm motility and progressive motility scores from cauda epididymis

193 under different transport conditions.


194 These results (shown in Figure 4, Table S3 and S4) showed that there were no significant

195 differences in spermatozoa motility in Group I, II and IV, but in Group III the spermatozoa

196 motility were higher when the cauda epididymis were transported at 4-8 oC than at RT regardless

197 of transport medium. These results of sperm progressive motility scores (Figure 4, Table S5 and

198 S6) showed that there were significant differences in Groups I, III and IV (p<0.05) except for

199 Group II. In Group II, the absence of transport medium did not significantly affect the sperm

200 progressive motility scores from transported cauda epididymis at 4-8 oC (p>0.05). However, no

201 matter under room temperature or at 4-8 oC, the absence of M2 medium will not affect the

202 motility and progressive motility scores of the transported spermatozoa in cauda epididymis

203 (p>0.05). It suggests that regardless of medium, temperature is the key factor of affecting sperm

204 progressive motility scores for transported cauda epididymis.

205 Differences in IVF from fresh and transported cauda epididymal sperm

206 We then examined the fertilization ability of fresh and the transported spermatozoa in cauda

207 epididymis at different transport conditions.

208 Fresh and transported spermatozoa in cauda epididymis were capacitated for 30 min at 37 oC and

209 then subjected to IVF (Table 1). These results revealed that the fertilization rates between fresh

210 and transported spermatozoa from cauda epididymis significantly differed when cauda

211 epididymis were transported at RT, but the fertilization rate of spermatozoa transported in cauda

212 epididymis at 4-8 oC was much better than spermatozoa transported at room temperature.

213 However, their subsequent blastocyst and birth rates of fertilized oocytes were not affected.

214 Effects of different transport temperatures and transport medium-free on fertilization rates and

215 the development of IVF embryos

216 Spermatozoa from the transported cauda epididymis were capacitated for 30 min at 37 oC and


217 then subjected to IVF (Table 2). These results suggested that the fertilization rates of the

218 transported spermatozoa in cauda epididymis significantly differed when transported at 4-8 oC

219 and at RT, regardless of the presence of M2. Furthermore, the fertilization rates of the

220 spermatozoa transported in cauda epididymis at 4-8 oC were considerably higher than those of

221 the spermatozoa transported at RT.

222 In order to clarify whether the presence or absence of transport medium affected the fertilization

223 rates of spermatozoa transported in cauda epididymis and the subsequent development of IVF

224 embryos from these spermatozoa, we summarized the data from the second experiment and

225 compared the fertilization rates and the embryonic development (Table 3).

226 Fertilization, blastocyst, and birth rates of the transported sperm via cauda epididymis in

227 transport medium at RT were 38%, 51.2%, and 42.9%, respectively; the corresponding rates of

228 the transported sperm in cauda epididymis without M2 at RT were 31.3%, 57.7%, and 41.5%;

229 the rates for sperm transported in cauda epididymis at 4-8 oC in M2 were 88.9%, 54.8%, and

230 40.2%, respectively; and the rates for sperm transported in cauda epididymis at 4-8 oC without

231 M2 were 85.9%, 58.5%, and 41%, respectively. These results suggested that there were no

232 significant differences in the fertilization rate and development of IVF embryos when the cauda

233 epididymis were transported at the same transport temperature regardless of transport medium.

234 However, the fertilization rate but not the embryo development of IVF was significantly affected

235 when the cauda epididymis were transported at RT comparing to 4-8°C, regardless of the

236 presence of transport medium or not.

237 Discussion

238 Many studies have reported the effects of the short-term storage of mouse sperm at RT or under

239 refrigeration. These findings provide useful information for optimalizing the conditions required


240 to maintain the motility and fertility of mouse sperm. A previous study demonstrated that mouse

241 sperm preserved in TYH medium (Toyoda 1971) at 24 oC could maintain their fertility for 24 h

242 (Jishage & Suzuki 1993). However, it is rather difficult to maintain sperm viability and normal

243 fertility for over 24 h at RT. Another study specifically evaluated the effect of M2, PB1, M16,

244 CZB, and TYH media on sperm motility, and M2 was found to be the most suitable among these

245 media for sperm storage at 22 oC (Sato & Ishikawa 2004), and M2 medium reported to be

246 optimal for maintaining high motility of stored sperm (Sato & Ishikawa 2004). Recently, Takeo

247 et al. (Takeo et al. 2012) compared the effects of paraffin oil, M2 medium, CPS-1, and Lifor on

248 sperm fertility after cold storage. Lifor was found to be superior to both M2 medium and paraffin

249 oil for maintaining the fertility of sperm collected from epididymides after 72 h of cold storage.

250 Therefore, the results from previous studies implied that the researchers were striving to discover

251 a more suitable medium but no one has found an optimal solution for sperm storage or transport

252 so far.

253 After maturation, spermatozoa are stored at the terminal region of the cauda epididymis for a

254 long period until ejaculation. Cauda epididymal spermatozoa have the capacity of progressive

255 motility but they are immotile (known as sperm quiescence) while suspended in native fluid. The

256 state of sperm quiescence and the lower temperature of the cauda epididymis (Djakiew &

257 Cardullo 1986) are advantageous as they enable the spermatozoa to conserve the energy required

258 to traverse the female genital tract and to fertilize the ovum. Kaneko et al. (Kaneko et al. 2009a)

259 suggested that short-term preservation of sperm without freezing could help avoid the marked

260 decrease in sperm motility and fertility that occurs due to freezing, and have found that the

261 spermatozoa preserved or transported in cauda epididymides at 4 oC could efficiently produce

262 offspring. Thus, the fertility of preserved or transported sperm in cauda epididymides at 4 oC


263 could be maintained, although sperm motility tended to gradually decrease with storage.

264 In order to exclude the effect of medium on sperm during storage and transport, and prevent the

265 leakage of epididymis fluid and sperm out of caua epididymis, in our present study we ligated

266 the both ends of caua epididymis completely for preventing the sample from drying due to loss

267 of moisture during transport. Therefore we used cauda epididymis as a carrier of sperm after they

268 were ligated at both ends and delivered them at 4-8 °C or RT.

269 We found that sperm fertility parameters in spermatozoa transported via cauda epididymides

270 were fairly good comparing with fresh sperm except for the caua epididymides transported at RT

271 (Table 1, p<0.05), although sperm motility was significantly different between fresh sperm and

272 those transported via cauda epididymides at 4-8 oC (Figure 3). Similarly, the spermatozoa

273 progressive motility scores of cauda epididymides that were transported at RT was inferior to

274 that of fresh spermatozoa(Figure 3). It suggests that the effect of room temperature on sperm

275 motility could ultimately lead to the decrease of fertilization rate.

276 Furthermore, among spermatozoa transported via the cauda epididymides with M2, those that

277 were transported at 4-8°C had a higher spermatozoa motility than those transported at RT (Figure

278 4, p<0.05), and although the presence or absence of M2 did not affect sperm progressive motility

279 scores at 4-8 oC, sperm motility and progressive motility score were negatively impacted when

280 transported at RT (Figure 4, p<0.05).

281 Although the fertilization rates of the transported spermatozoa in cauda epididymis at the same

282 temperature were comaparably same, we eventually found that the fertilization rates of the

283 transported sperm in cauda epididymis at 4-8 oC were much higher than that at RT. It suggested

284 that in the process of cauda epididymis or sperm transport, temperature was more critical to

285 maintain sperm quality and its fertility(Table 2). Our results suggest that during sperm transport


286 via cauda epididymides, absence of transport medium influenced the fertility much less than

287 transport temperature, and the efficiency of RT transport was always not better than that of 4-8

288 oC.

289 Spermatozoa transported at RT with and without M2 yielded IVF fertilization rates of 38% and

290 31.3%, respectively (Table 3). Moreover, the two-cell embryos from the sperm transported at RT

291 without transport medium could develop to blastocysts at higher developmental rates (57.7%),

292 and healthy pups were obtained from these sperm samples after embryonic transfer,

293 demonstrating the feasibility of sperm delivery at room temperature without transport medium.

294 There are several advantages of sperm transport at room temperature or 4-8 oC as follows: (i) it

295 does not require special containers such as dry shippers, (ii) it does not need bacteriological

296 monitoring for delivery registration, (iii) it does not require the thawing step for subsequent in

297 vitro fertilization and (iv) there is no frostbite risk for the operator as the frozen/transported

298 spermatozoa in liquid nitrogen (LN2) tank. Moreover this transport equipment is cost-effective,

299 easy to assemble and carry. Additionaly, if season temperature is suitable (4-25oC), this mode of

300 transport should be more convenient and simple comparing with LN2 transport. Epididymis

301 samples just need simple packaging to achieve the purpose of transporting sperm at a high

302 efficiency.

303 In summary, our findings suggest that transport of spermatozoa via the cauda epididymidis at 4-8

304 oC can help maintain a high level of fertility of the transported spermatozoa as compared to

305 transport at RT; furthermore, the modified transport medium-free system is more beneficial to

306 transport of spermatozoa via cauda epididymides at 4-8 oC than at room temperature. Therefore,

307 we strongly believe that our modifications could improve the usefulness of cauda epididymis

308 transport at 4-8 oC. Moreover, we also proved the feasibility of sperm transport at RT although


309 the fertilization capacity of these sperm were sightly poor.

310 Acknowledgments

311 This study was supported by Science and Technology Planning Project of Guangdong Province,

312 China (No. 2014A020213025). We thank Drs Peng-Cheng Kong, Shen Li, Lei-Ning Chen and

313 Ms Shao-Qing Chen for their technical expertises and valuable advice during study and

314 manuscript writing.

315 Reference

316 An TZ, Wada S, Edashige K, Sakurai T, and Kasai M. 1999. Viable spermatozoa can be recovered from refrigerated 317 mice up to 7 days after death. Cryobiology 38:27-34. S0011-2240(98)92141-1 [pii]318 10.1006/cryo.1998.2141319 Batzer FR, Hurwitz JM, and Caplan A. 2003. Postmortem parenthood and the need for a protocol with posthumous 320 sperm procurement. Fertil Steril 79:1263-1269. S0015028203003844 [pii]321 Belker AM, Swanson ML, Cook CL, Carrillo AJ, and Yoffe SC. 2001. Live birth after sperm retrieval from a 322 moribund man. Fertil Steril 76:841-843. S0015-0282(01)02013-1 [pii]323 Check M, Summers-Chase D, Check JH, Choe J, and Nazari A. 1999. Sperm extracted and cryopreserved from 324 testes several hours after death results in pregnancy following frozen embryo transfer: case report. Arch 325 Androl 43:235-237. 326 Djakiew D, and Cardullo R. 1986. Lower temperature of the cauda epididymidis facilitates the storage of sperm by 327 enhancing oxygen availability. Gamete Research 15:237-245. 10.1002/mrd.1120150305328 Fuller SJ, and Whittingham DG. 1996. Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and 329 embryonic development. J Reprod Fertil 108:139-145. 330 Jequier AM, and Crich JP. 1986. Semen analysis: a practical guide: Blackwell Scientific Publications; St. Louis, 331 Mo.: Distributors, USA, Blackwell Mosby Book Distributors.332 Jishage K, and Suzuki H. 1993. Maintenance of the Fertilizing Ability in Capacitated Mouse Spermatozoa. Journal 333 of Reproduction and Development 39:363-367. 334 Jishage K, Ueda O, and Suzuki H. 1997. Fertility of mouse spermatozoa from cauda epididymis preserved in 335 paraffin oil at 4 C. J Mamm Ova Res 14:45-48. 336 Kaneko T, Fukumoto K, Haruguchi Y, Kondo T, Machida H, Koga M, Nakagawa Y, Tsuchiyama S, Saiki K, and 337 Noshiba S. 2009a. Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after 338 preservation or transportation at 4 C using laser-microdissected oocytes. Cryobiology 59:59-62. 339 Kaneko T, Fukumoto K, Haruguchi Y, Kondo T, Machida H, Koga M, Nakagawa Y, Tsuchiyama S, Saiki K, 340 Noshiba S, and Nakagata N. 2009b. Fertilization of C57BL/6 mouse sperm collected from cauda 341 epididymides after preservation or transportation at 4 degrees C using laser-microdissected oocytes. 342 Cryobiology 59:59-62. 10.1016/j.cryobiol.2009.04.006343 S0011-2240(09)00040-6 [pii]344 Mochida K, Ohkawa M, Inoue K, Valdez DM, Jr., Kasai M, and Ogura A. 2005. Birth of mice after in vitro 345 fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures. 346 Theriogenology 64:135-143. S0093-691X(04)00403-0 [pii]347 10.1016/j.theriogenology.2004.11.013348 Pease S, Saunders TL, Sztein JM, Kastenmayer RJ, and Perdue KA. 2011. Pathogen-Free Mouse Rederivation by 349 IVF, Natural Mating and Hysterectomy. Advanced Protocols for Animal Transgenesis: Springer Berlin 350 Heidelberg, 615-642.351 Rothman CM. 1980. A method for obtaining viable sperm in the postmortem state. Fertil Steril 34:512. 352 Sankai T, Tsuchiya H, and Ogonuki N. 2001. Short-term nonfrozen storage of mouse epididymal spermatozoa. 353 Theriogenology 55:1759-1768. S0093-691X(01)00518-0 [pii]354 Sato M, and Ishikawa A. 2004. Room temperature storage of mouse epididymal spermatozoa: exploration of factors


355 affecting sperm survival. Theriogenology 61:1455-1469. 10.1016/j.theriogenology.2003.07.013356 S0093691X03003078 [pii]357 Sato M, Ishikawa A, Nagashima A, Watanabe T, Tada N, and Kimura M. 2001. Prolonged survival of mouse 358 epididymal spermatozoa stored at room temperature. Genesis 31:147-155. 10.1002/gene.10011 [pii]359 Takeo T, Tsutsumi A, Omaru T, Fukumoto K, Haruguchi Y, Kondo T, Nakamuta Y, Takeshita Y, Matsunaga H, 360 Tsuchiyama S, Sakoh K, Nakao S, Yoshimoto H, Shimizu N, and Nakagata N. 2012. Establishment of a 361 transport system for mouse epididymal sperm at refrigerated temperatures. Cryobiology 65:163-168. 362 10.1016/j.cryobiol.2012.06.002363 S0011-2240(12)00113-7 [pii]364 Toyoda Y. 1971. Studies on fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal 365 sperm. Jpn J Anim Reprod 16:147-151.


367 Tables and Figure Legends

368 Table 1. Comparison of the fertilization rates between fresh sperm and the sperm

369 fromtransported caudal epididymides and the subsequent development of IVF embryos. *RT,

370 room temperature; “-”, without transport medium; “+”, with transport medium. Values with

371 different superscripts within columns are significantly different at P < 0.05 (Chi-square test).

372 Table 2. Comparison of the fertilization rates of the sperm from transported caudal epididymides

373 at different conditions and the subsequent development of IVF embryos. *RT, room temperature;

374 “-”, without transport medium; “+”, with transport medium.Values with different superscripts

375 within columns are significantly different at P < 0.05 (Chi-square test).

376 Table 3. Summary of the fertilization rates of the sperm from transported caudal epididymides at

377 different conditions and the development of IVF embryos. *RT, room temperature; “-”, without

378 transport medium; “+”, with transport medium. Values with different superscripts within

379 columns are significantly different at P < 0.05 (Chi-square test).

380 Table S1. Raw Data of Spermatozoa Motility from Experiment 1. RT, room temperature; “-”,

381 without transport medium; “+”, with transport medium.

382 Table S2. Raw Data of Spermatozoa Progressive Motility Scores from Experiment 1. RT, room

383 temperature; “-”, without transport medium; “+”, with transport medium.

384 Table S3. Raw Data of Spermatozoa Mobility from Experiment 2. RT, room temperature; “-”,

385 without transport medium; “+”, with transport medium.

386 Table S4. Comparison Results of Spermatozoa Mobility from Experiment 2 by One-way Anova.

387 RT, room temperature; “-”, without transport medium; “+”, with transport medium. The asterisks

388 represent significant differences (t test, P < 0.05). NS menas no significance.

389 Table S5. Raw Data of Spermatozoa Progressive Mobility Scores from Experiment 2. RT, room


390 temperature; “-”, without transport medium; “+”, with transport medium.

391 Table S6. Comparison Results of Spermatozoa Progressive Mobility Scores from Experiment 2

392 by One-way Anova. RT, room temperature; “-”, without transport medium; “+”, with transport

393 medium. The asterisks represent significant differences (t test, P < 0.05). NS menas no

394 significance.

395

396 Figure 1. Design of Experiment 1.

397 One side (A) cauda epididymis was used for in vitro fertilization in each group (1 - 4). Another

398 side of tcauda epididymis obtained from the same mouse was ligated at both ends for transport at

399 RT (B, Group 1) or 4-8 oC (B, Group 2) with 20 μL of transport medium M2, or ligated at both

400 ends for transport at RT (B, Group 3) or 4-8 oC without M2 (B, Group 4). The cauda

401 epididymides were transported to the destination within 24 h of collection. Once cauda

402 epididymis arrived within 24 h after collection and met the acceptance criteria, spermatozoa were

403 released out of the samples for assessing sperm motility and progressive motility scores, and in

404 vitro fertilization.

405 Figure 2. Design of Experiment 2.

406 Two sides (A and B) of cauda epididymis from the same mouse were ligated at both ends and

407 transported at RT with or without 20 μL of M2 (Group I), at 4-8 oC with or without M2 (Group

408 II), with M2 at 4-8 oC or RT (Group III), and without M2 at 4-8 oC or RT (Group IV). Once

409 cauda epididymis arrived within 24 h after collection, spermatozoa were released from the

410 samples for assessing sperm motility and progressive motility scores, and in vitro fertilization.

411 Figure 3. Motility and progressive motility scores of fresh spermatozoa and sperm from

412 transported cauda epididymides at RT or 4-8 oC with or without M2.


413 Spermatozoa motility (upper panel) and progressive motility scores (lower panel) in BDF1

414 mouse spermatozoa after capacitation at 37 oC for 30 min. The spermatozoa were delivered via

415 the cauda epididymis at RT without M2 (Group 1), at 4-8 oC without M2 (Group 2), at RT with

416 M2 (Group 3), and at 4-8 oC with M2 (Group 4). Fresh sperm from the same mice were used as

417 controls in each group.

418 Error bars represent ± standard error of the mean (SEM). The asterisks on the bars represent

419 significant differences (t test, P < 0.05). NS menas no significance.

420 Figure 4. Motility and progressive motility scores of sperm from the transported cauda

421 epididymides at RT or 4-8 oC at the presence or absence of transport medium M2.

422 Spermatozoa motility (upper panel) and progressive motility scores (lower panel) in BDF1

423 mouse spermatozoa after capacitation at 37 oC for 30 min. The spermatozoa were delivered via

424 the cauda epididymis at RT with or without M2 (Group I), at 4-8 oC with or without M2 (Group

425 II), with M2 at 4-8 oC or RT (Group III), and without M2 at 4-8 oC or RT (Group IV).

426 Error bars represent ± SEM. Different letters on the bars represent significant differences (t test,

427 P < 0.05).

428


Figure 1(on next page)

Design of Experiment 1.

One side (A) cauda epididymis was used for in vitro fertilization in each group (1 - 4). Another side of tcaudaepididymis obtained from the same mouse was ligated at both ends for transport at RT (B, Group 1) or 4-8oC (B, Group 2) with 20 μL of transport medium M2, or ligated at both ends for transport at RT (B, Group 3)or 4-8 oC without M2 (B, Group 4). The cauda epididymides were transported to the destination within 24 hof collection. Once cauda epididymis arrived within 24 h after collection and met the acceptance criteria,spermatozoa were released out of the samples for assessing sperm motility and progressive motility scores,and in vitro fertilization.




Design of Experiment 2.

Two sides (A and B) of cauda epididymis from the same mouse were ligated at both ends and transported atRT with or without 20 μL of M2 (Group I), at 4-8 oC with or without M2 (Group II), with M2 at 4-8 oC or RT(Group III), and without M2 at 4-8 oC or RT (Group IV). Once cauda epididymis arrived within 24 h aftercollection, spermatozoa were released from the samples for assessing sperm motility and progressivemotility scores, and in vitro fertilization.




Motility and progressive motility scores of fresh spermatozoa and sperm fromtransported cauda epididymides at RT or 4-8 oC with or without M2.




Motility and progressive motility scores of sperm from the transported caudaepididymides at RT or 4-8 oC at the presence or absence of transport medium M2.



Table 1(on next page)

Comparison of the fertilization rates between fresh sperm and the spermfromtransported caudal epididymides and the subsequent development of IVF embryos.


Table 1. Comparison of the fertilization rates between fresh sperm and the sperm from transported caudal epididymides and the subsequent development of IVF

embryos

Group Side Sperm type* Replicates No. oocytes Percentage of

cleavage Percentage of

blastocysts No. transferred

blastocysts Pups born (%)

1 A B

Fresh RT (-) 3 98

118 92.9 (91/98)a 31.4(37/118)b

60.4(55/91)a 59.5(22/37)a

55 22

23(41.8)a 10(45.5)a

2 A B

Fresh 4-8°C (-) 3 89

108 94.4(84/89)a

86.1(93/108)a 57.1(48/84)a 58.1(54/93)a

48 54

21(43.8)a 22(40.7)a

3 A B

Fresh RT (+) 3 102

104 89.2(91/102)a 38.5(40/104)b

56.0(51/91)a 52.5(21/40)a

51 21

20(39.2)a 9(42.9)a

4 A B Fresh

4-8°C (+) 3 90 128

90.0(81/90)a 89.1(114/128)a

59.3(48/81)a 60.5(69/114)a

48 69

20(41.7)a 29(42.0)a

*RT, room temperature; “-”, without transport medium; “+”, with transport medium.

Values with different superscripts within columns are significantly different at P < 0.05 (Chi-square test).



Comparison of the fertilization rates of the sperm from transported caudal epididymidesat different conditions and the subsequent development of IVF embryos.


Table 2. Comparison of the fertilization rates of the sperm from transported caudal epididymides at different conditions and the subsequent development of IVF

embryos

Group Side Sperm type* Replicates Total No. oocytes Percentage of

cleavage Percentage of

blastocysts No. transferred

blastocysts Pups born (%)

I A B

RT (+) RT (-) 3 103

112 37.9(39/103)a 32.1(36/112)a

51.3(20/39)a 58.3(21/36)a

20 21

9(45.0)a 9(42.9)a

II A B

4-8°C (+) 4-8°C (-) 3 91

104 89(81/91)b

85.6(89/104)b 55.6(45/81)a 57.3(51/89)a

45 51

19 (42.2)a 22(43.1)a

III A B

4-8°C (+) RT (+) 3 98

113 88.8(87/98)b 38.1(43/113)a

54(47/87)a 51.2(22/43)a

47 22

18(38.3)a 9(40.9)a

IV A B 4-8°C (-)

RT (-) 3 95 115

86.3(82/95)b 30.4(35/115)a

59.8(49/82)a 57.1(20/35)a

49 20

19(38.8)a 8(40.0)a





Summary of the fertilization rates of the sperm from transported caudal epididymides atdifferent conditions and the development of IVF embryos.


Table 3. Summary of the fertilization rates of the sperm from transported caudal epididymides at different conditions and the development of IVF embryos

Sperm type* Replicates Total No. oocytes Percentage of cleavage

Percentage of blastocysts

No. transferred blastocysts Pups born (%)

RT (+) RT(-) 4-8℃ (+) 4-8℃ (-)

6 6 6 6

216 227 189 199

38(82/216)a 31.3(71/227)a

88.9(168/189)b 85.9(171/199)b

51.2(42/82)a 57.7(41/71)a

54.8(92/168)a 58.5(100/171)a

42 41 92

100

18(42.9)a 17(41.5)a 37 (40.2)a 41(41.0)a




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