1 1

1 THE SUPEROXIDE DISMUTASE MIMETIC MnTBAP DECREASES ROS

2 PRODUCTION AND MAINTAINS VIABILITY DURING

3 CRYOPRESERVATION OF HUMAN SPERMATOZOA

4
5
6 Pamela Uribe1,2, Constanza Cárcamo1, Eliana Navarro1, Josefa

7 Sepúlveda1, Fabiola Zambrano1,3, Mabel Schulz1,3 and Raúl Sánchez1,3*.

8
1
9 Center of Excellence in Translational Medicine ‐ Scientific and

10 Technological Bioresource Nucleus (CEMT – BIOREN), 2Department of

3
11 Internal Medicine, Department of Preclinical Sciences, Faculty of

12 Medicine, Universidad de La Frontera, Temuco, Chile.

13

14 *Correspondence address: Postal address: Avenida Alemania 0458,

15 Temuco - Chile; Tel: +56-45-2596584; E-mail: raul.sanchez@ufrontera.cl

16

17

18 Running title: Use of MnTBAP in human sperm vitrification

19

20 Key words: Sperm cryopreservation, sperm vitrification, human sperm,

21 reactive oxygen species, oxidative stress

22

23

24
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25 ABSTRACT

26 Assisted reproduction technologies and gamete cryopreservation

27 play an important role in the treatment of male infertility. Sperm

28 cryopreservation is widely used in assisted reproduction procedures;

29 however, it induces oxidative stress affecting sperm quality, therefore, the

30 use of antioxidants can improve cryopreservation outcome. The objective

31 here was to evaluate the effect of the supplementation of vitrification,

32 devitrification and incubation post-devitrification media with the antioxidant

33 MnTBAP on the quality of cryopreserved human sperm. First, the effect of

34 MnTBAP on sperm viability and reactive oxygen species (ROS) was

35 evaluated. Then, the spermatozoa were cryopreserved by the vitrification

36 technique with the vitrification, devitrification and incubation post-

37 devitrification media separately supplemented with MnTBAP. An untreated

38 control was included. The sperm viability, ROS production, total and

39 progressive motility were evaluated. The results showed that the

40 supplementation of the vitrification medium did not affect the parameters

41 analyzed. However, the supplementation of the devitrification and

42 incubation post-devitrification media resulted in a decrease in ROS

43 production with maintained viability. Only the higher concentration of

44 MnTBAP caused a decrease in total motility after 4 hours of incubation

45 post-devitrification. In conclusion, the use of MnTBAP during vitrification

46 has a beneficial effect, decreasing ROS production in human sperm.

47
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48 INTRODUCTION

49

50 Infertility currently affects approximately 15% of couples worldwide

51 and every year more than seven million couples need help for fertility

52 success (Pan et al., 2018). In this context, assisted reproduction

53 technologies (ARTs) and gamete cryopreservation play an important role

54 in the treatment of infertility (Agarwal et al., 2014). Sperm cryopreservation

55 aims to keep cells at low temperatures for long periods of time,

56 maintaining their fertilizing capacity (Di Santo et al., 2012). Although

57 cryopreservation is widely used in assisted reproduction procedures, there

58 is sub-lethal damage to cryopreserved spermatozoa, which results in

59 deteriorated fertilization ability (Ortega Ferrusola et al., 2009). This

60 damage may be associated with oxidative stress generated by the

61 increased production of reactive oxygen species (ROS), which occurs as a

62 result of cryopreservation and the incubation of the spermatozoa after

63 cryopreservation (Uribe et al., 2017). As a strategy to minimize the

64 damage produced by elevated ROS levels, the use of antioxidants during

65 cryopreservation protocols has been proposed, which could contribute to

66 maintaining sperm quality after cryopreservation (Amidi et al., 2016). One

67 alternative is the antioxidant MnTBAP manganese (III) meso-tetrakis (4-

68 benzoic acid) porphyrin, a cell-permeable synthetic compound that

69 belongs to a group of metalloporphyrin antioxidants, which are

70 characterized by a redox-active transitional metal (Mn or Fe) coordinated

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71 to a cyclic porphyrin core ligand (Sheng et al., 2014). Metalloporphyrins

72 have emerged as a class of therapeutic catalytic antioxidants that

73 scavenge a wide range of ROS and reactive nitrogen species, and several

74 compounds in this class have been shown to be efficacious in a variety of

75 in vitro and in vivo oxidative stress models of human diseases for an

76 extensive review see (Patel and Day, 1999). The beneficial effects of

77 MnTBAP are attributed to a powerful antioxidant action on the cytoplasm

78 and mitochondrion of the cell, since, in addition to turning O 2- into H2O2, it

79 catalyzes the dissociation of H 2O2 in water, blocking the damage produced

80 by ROS (Forouzanfar et al., 2013; Shojaeian et al., 2018). Accordingly, the

81 supplementation of semen extender with this antioxidant improved sperm

82 quality parameters after thawing in stallion (Shojaeian et al., 2018; Treulen

83 et al., 2019) and ram sperm (Forouzanfar et al., 2013); however, there are

84 currently no reports that demonstrate the effect of MnTBAP on the

85 cryopreservation of human spermatozoa. In addition, most studies that

86 evaluate the antioxidant effect during cryopreservation protocols only

87 analyze the supplementation of freezing media; however, ROS

88 overproduction could also occur during thawing or post-thaw incubation

89 (Uribe et al., 2017). With this background, the objective here was to

90 evaluate the effect of the supplementation of vitrification, devitrification and

91 incubation post-devitrification media with the antioxidant MnTBAP on the

92 quality of cryopreserved human sperm.

93
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94 MATERIAL AND METHODS

95 Semen samples

96 Evaluations were made with spermatozoa obtained from semen

97 samples of normozoospermic healthy donors. This study was approved by

98 the Scientific Ethics Committee of the Universidad de La Frontera and

99 each donor signed an informed consent.

100 The semen samples were obtained by masturbation after at least 3

101 days of sexual abstinence, collected in sterile containers and delivered

102 immediately to the laboratory. The swim up method was used to select the

103 motile spermatozoa from the semen samples.

104

105 Experimental design

106 Analysis of the effect of MnTBAP in human spermatozoa

107 First, the dosage effect of MnTBAP on human spermatozoa was

108 evaluated in order to establish the antioxidant concentrations to use in the

109 subsequent cryopreservation experiments. To do this, human

110 spermatozoa (3 × 106 cells/ml) were exposed to concentrations ranging

111 from 0 (control), 50, 100 150 and 200 µmol/l of MnTBAP at 37°C for one

112 hour. After incubation, the spermatozoa were washed twice with human

113 tubular fluid (HTF) medium by centrifugation at 500 xg for 5 minutes. ROS

114 production and viability were assessed by incubation at 37 ºC for 15

115 minutes with 2 mol/l of dihydroethidium (DHE, Molecular Probes,

116 ThermoFisher, MA, USA) in combination with 25 nmol/l of SYTOX Green

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117 (Molecular Probes, ThermoFisher, MA, USA). After incubation, the

118 spermatozoa were washed once and re-suspended in 300 µl of HTF for

119 analysis by flow cytometry (See Analysis by flow cytometry, below).

120

121 Cryopreservation procedure

122 Next, spermatozoa were cryopreserved with the different media

123 separately supplemented with MnTBAP. The cryopreservation procedure

124 applied was aseptic vitrification, following the previously described

125 protocol (Isachenko et al., 2012; Uribe et al., 2017). The vitrification

126 solution consisted of Sydney IVF Gamete Buffer (Cook Medical Inc.

127 Bloomington, Indiana, USA) supplemented with 0.25 mol/l sucrose

128 (Sigma-Aldrich Inc. St Louis, MO, USA) and with 1% Dextran Serum

129 Supplement (Irvine Scientific, Santa Ana, California, USA). Spermatozoa

130 were centrifuged and re-suspended with vitrification solution to achieve a

131 sperm concentration of 30 × 106 cells/ml. Then, 100 μl of vitrification

132 solution containing spermatozoa were placed in a 0.25 ml straw, which

133 was introduced into a 0.5 ml straw, sealed by heat at both ends and

134 immediately plunged into liquid nitrogen, where they were stored until

135 devitrification and analysis. For devitrification, the 0.25 ml straw containing

136 the vitrified spermatozoa was immersed in a 15 ml polypropylene tube

137 containing 2 ml of devitrification medium (Sydney IVF Gamete Buffer

138 supplemented with 1% Dextran Serum Supplement), which was pre-

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139 warmed to 42 °C. The spermatozoa were washed by centrifugation for 5

140 minutes at 700 RCF and re-suspended with HTF medium for analysis.

141

142 Effect of the supplementation of vitrification medium with MnTBAP

143 For experiments including MnTBAP in the vitrification procedure,

144 first spermatozoa were vitrified with vitrification medium supplemented

145 with different concentrations of MnTBAP 0 (control), 50, 100 and 150

146 µmol/l. Once devitrified, the spermatozoa were washed twice with HTF

147 and the sperm viability and ROS production were evaluated as previously

148 described. In addition, sperm motility was analyzed with the CASA

149 (computer-assisted sperm analysis) system using the SCA production

150 software (MICROPTIC S.L., Barcelona, Spain).

151

152 Effect of the supplementation of devitrification medium with MnTBAP

153 Then, experiments were conducted to evaluate the effect of the

154 supplementation of devitrification medium with MnTBAP 0 (control), 50,

155 100 and 150 µmol/l. In this experiment the vitrification medium was not

156 supplemented. Once devitrified, the spermatozoa were washed 3 times by

157 centrifugation for 5 minutes at 700 RCF and the viability, ROS production

158 and motility were analyzed as previously described.

159

160 Effect of the supplementation of incubation post-devitrification medium

161 with MnTBAP

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162 Finally, the effect of the supplementation of incubation post-

163 devitrification medium with MnTBAP was evaluated. To do this, according

164 to the results obtained in the previous experiments, the vitrification

165 medium was not supplemented with MnTBAP, whereas the devitrification

166 medium was supplemented with 100 µmol/l of MnTBAP and the post-

167 devitrification incubation medium was supplemented with different

168 concentrations of MnTBAP 0 (control), 50, 100 and 150 µmol/l. Post-

169 devitrification incubation was done at room temperature because the

170 sperm parameters are better maintained than incubation at 37 ºC (Uribe et

171 al., 2017). Once incubation was complete, the spermatozoa were washed

172 3 times and the viability, ROS production and motility were analyzed as

173 previously described.

174

175 Analysis by flow cytometry

176 The fluorescent staining analyses were performed in a FACSCanto

177 II flow cytometer controlled by the FACSDiva™ v. 6.1.3 software (Becton,

178 Dickinson and Company, BD Biosciences, San Jose, CA, USA). Excitation

179 of fluorochromes was performed with a 488-nm argon laser. The

180 fluorescence from SYTOX Green was detected using the band pass filter

181 of 530/30 nm, and the fluorescence from DHE with a band pass filter of

182 585/42 nm. All analyses were done on logarithmic scales and data from

183 10,000 sperm events were analyzed in each experiment.

184
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185 Statistical analysis

186 All the parameters in each experimental group and controls were

187 evaluated in duplicate, and the experiments were repeated at least 3 times

188 on different days and with samples from different donors. The results were

189 expressed as the mean ± standard deviation. For the statistical evaluation

190 the Prism 5 software (GraphPad, La Jolla, CA, USA) was used. To verify

191 the normal distribution of the data, D’Agostino’s K2 test was applied and

192 the numerical results were transformed to a logarithmic scale when they

193 did not pass the test of normality. For the comparison of the experimental

194 groups vs. their respective control a one-way analysis of variance

195 (ANOVA) and Dunnett’s post-test was applied. Differences were

196 considered statistically significant for values p<0.05.

197

198 RESULTS

199

200 Effect of MnTBAP in human spermatozoa

201 The results regarding the effect of MnTBAP in sperm cells showed

202 that the viability was not altered with concentrations up to 200 µmol/l of

203 MnTBAP in comparison with the untreated control (Figure 1A). The ROS

204 levels in the spermatozoa decreased significantly with all the MnTBAP

205 concentrations studied in comparison with the untreated control (Figure

206 1B), indicating an effective activity of MnTBAP decreasing ROS levels.

207 According to these results and what was described previously (Treulen et
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208 al., 2019), concentrations of 50, 100 and 150 µmol/l of MnTBAP were

209 used for the supplementation of the vitrification, devitrification and post-

210 devitrification incubation media.

211

212 Effect of the supplementation of vitrification medium with MnTBAP

213 When spermatozoa were cryopreserved with vitrification medium

214 supplemented with MnTBAP the results showed that viability, ROS

215 production and total or progressive motility were not different in

216 comparison with the untreated control (Table I, supplementation of

217 vitrification medium).

218

219 Effect of the supplementation of devitrification medium with MnTBAP

220 When the devitrification medium was supplemented with MnTBAP

221 the sperm viability and the total and progressive motility were maintained,

222 whereas the ROS production was significantly lower when the medium

223 was supplemented with 100 µmol/l of MnTBAP in comparison with the

224 untreated control (Table I, supplementation of devitrification medium).

225

226 Effect of the supplementation of incubation post-devitrification medium

227 with MnTBAP

228 Finally, the post-devitrification incubation medium was

229 supplemented with MnTBAP and, considering the previous results, in this

230 experiment the devitrification medium was also supplemented with 100
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231 µmol/l of MnTBAP. The devitrified spermatozoa kept for 4 hours at room

232 temperature with the incubation medium supplemented with MnTBAP did

233 not show any alteration in sperm viability (Table I, supplementation of

234 incubation post-devitrification medium). Moreover, the supplementation of

235 incubation medium with 50 µmol/l of MnTBAP caused a significant

236 reduction in ROS production compared to the control of spermatozoa

237 incubated in the medium without MnTBAP supplementation (Table I,

238 supplementation of incubation post-devitrification medium). However, the

239 total motility decreased significantly at 4 hours of incubation with 150

240 µmol/l of MnTBAP, while the progressive motility was not affected by any

241 of the concentrations studied (Table I, supplementation of incubation post-

242 devitrification medium).

243

244 DISCUSSION

245 Sperm cryopreservation is essential in ARTs and male fertility

246 preservation (Li et al., 2019); however, this procedure promotes oxidative

247 stress in sperm cells as it induces excessive production of ROS (Karimfar

248 et al., 2015; Mazzilli et al., 1995; Wang et al., 1997), which can also be

249 generated by incubation of sperm cells after cryopreservation (Uribe et al.,

250 2017). Oxidative stress is well known to cause alterations at several levels

251 in sperm cells, decreasing their fertilizing ability (Dutta et al., 2019;

252 Gharagozloo and Aitken, 2011). Successful strategies aimed at reducing

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253 the oxidative damage generated during the cryopreservation process

254 would improve the cryopreservation outcome.

255 In this study, it was demonstrated that the supplementation of the

256 devitrification and incubation post-devitrification medium with up to 100

257 mol/l of MnTBAP reduces ROS production, maintaining viability in human

258 spermatozoa. Our results agree with previous reports indicating that

259 MnTBAP as a supplement of the semen extender improved sperm motility

260 and viability, while it decreased the lipid peroxidation and DNA damage in

261 stallion sperm (Shojaeian et al., 2018; Treulen et al., 2019). Also similar to

262 our results, in ram sperm the supplementation of the extender with

263 MnTBAP resulted in higher sperm motility and improved acrosomal

264 membrane integrity, with 100 mol/l being the optimum MnTBAP

265 concentration (Forouzanfar et al., 2013).

266 Interestingly, the supplementation with the antioxidant of

267 devitrification and incubation post-devitrification media but not the

268 supplementation of vitrification medium decreased sperm ROS production,

269 which suggests the greatest ROS production in human spermatozoa

270 occurs during the devitrification and post-devitrification incubation process.

271 This is useful to consider in future studies designed to evaluate the use of

272 antioxidants during sperm cryopreservation protocols, since most of the

273 studies report the effect of the antioxidants included only in the freezing or

274 vitrification medium.

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275 In addition, a previous study by our research group demonstrated

276 that vitrification induces a significant increase in ROS production in the

277 spermatozoa and that these reactive species continue increasing in a

278 time-dependent manner when the devitrified spermatozoa are kept at

279 room temperature or at 37 ºC (Uribe et al., 2017). Here, we reported that

280 the addition of MnTBAP to post-devitrification incubation medium makes it

281 possible to maintain spermatozoa with a lower ROS production than the

282 spermatozoa incubated without MnTBAP supplementation. These results

283 can help optimize the handling protocols for devitrified spermatozoa,

284 particularly those that will be used in assisted reproduction techniques,

285 where the best possible sperm quality is necessary.

286

287 ACKNOWLEDGEMENTS

288 This work was supported by the the Comisión Nacional de

289 Investigación Científica y Tecnológica, Chile [Grant number PAI79160030

290 to P.U.], CONICYT, Fondo Nacional de Investigación Científica y

291 Tecnológica (FONDECYT), Chile [Grant number 11170758 to P.U.] and

292 Vicerrectoría de Investigación y Postgrado, Dirección de Investigación,

293 Universidad de La Frontera, Temuco, Chile.

294

295 CONFLICT OF INTEREST

296 The authors declare no conflict of interest.

297
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298 AUTHORS’ CONTRIBUTION

299 PU: study design, data analysis and interpretation, drafting of the

300 article and approval of the submitted and final version. CC, EN, JS: data

301 acquisition and analysis, drafting of the article and approval of the

302 submitted and final version. FZ, MS and RS: data analysis and

303 interpretation, critical revision of the article and approval of the submitted

304 and final version.

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365 Shojaeian K, Nouri H, Kohram H (2018) Does MnTBAP ameliorate DNA fragmentation
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395 FIGURE LEGEND

396

397 Figure 1. Effect of MnTBAP on human spermatozoa. The spermatozoa

398 were exposed to different concentrations of MnTBAP for 1 hour at 37°C,

399 including an untreated control (0 mol/l of MnTBAP). Viability (A) and the

400 production of reactive oxygen species (B) were analyzed. The results are

401 expressed as the average ± SD of 5 different experiments. (*) p < 0.05;

402 (***) p < 0.001 compared to the untreated control. Abbreviations: ROS:

403 reactive oxygen species, MFI: mean fluorescence intensity, AU: arbitrary

404 units.

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418 TABLE I
419
420 Table I. Effect of suplementation of vitrification, devitrification and

421 incubation post-devitrification media with the antioxidant MnTBAP.

422

Supplementation of vitrification medium

MnTBAP Viability (%) ROS production Total motility Progressive

(mol/l) (%) motility (%)
(MFI, AU)

0 69.3  9.7 2150  774.2 55.0  26.2 47.3  22.8

50 62.5  11.6 2603  995.6 58.0  30.7 52.5  30.1

100 66.8  8.3 2313  691.3 47.0  28.3 39.7  26.8

150 66.6  7.7 2169  500.5 47.4  26.8 40.3  23.4

Supplementation of devitrification medium

0 70.5  8.1 2136  526.4 58.4  21.2 51.2  18.7

50 73.8  9.1 1778  536.8 55.3  27.2 45.1  21.4

100 75.3  5.2 1727  388.8 * 57.5  22.3 46.8  20.5

150 71.9  8.0 1977  491.5 52.0  23.8 45.3  22.3

Supplementation of incubation post-devitrification medium

0 78.0  7.8 1488  441.5 61.6  13.3 47.2  13.2

50 79.7  1.8 1090  169.1 * 50.8  25.0 37.2  24.7

100 76.0  4.4 1149  154.6 48.7  16.3 38.0  14.4

150 76.3  5.3 1129  159.7 38.2  18.1 * 31.5  17.2

423
424 Values correspond to mean  SD after cryopreservation of 5
425 (supplementation of vitrification and devitrification media) and 3
426 (supplementation of post-devitrification medium) different experiments.
427 (*) p < 0.05 compared to the untreated control (0 mol/l MnTBAP).
428 ROS: reactive oxygen species, MFI: mean fluorescence intensity, AU:
429 arbitrary units.
430