YCRYO 3608 No.

of Pages 6, Model 5G
22 May 2015

Cryobiology xxx (2015) xxx–xxx

1

Contents lists available at ScienceDirect

Cryobiology
journal homepage: www.elsevier.com/locate/ycryo

4
5

3 Effects of vitrification on ram spermatozoa using free-egg yolk extenders

6 Pilar Jiménez-Rabadán a,⇑, Olga García-Álvarez b, Ana Vidal b, Alejandro Maroto-Morales b,
7 María Iniesta-Cuerda b, Manuel Ramón a, Enrique del Olmo b, Rocío Fernández-Santos b, J. Julián Garde b,
8 Ana Josefa Soler b
9 a
Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain
10 b
SaBio (IREC) CSIC-UCLM-JCCM, 02071 Albacete, Spain

11
a r t i c l e i n f o a b s t r a c t
1
2 3
5
14 Article history: The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process 26
15 Received 4 December 2014 in free-egg-yolk diluents in relation with conventional diluents and cryopreservation protocol used in 27
16 Accepted 13 May 2015 this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based 28
17 Available online xxxx
on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% 29
and 18%) compared to a commercial extender (BiladylÒ with 20% egg-yolk and 7% glyerol). 30
18 Keywords: Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were 31
19 Vitrification
compared with the conventional cryopreservation. Semen from three rams was collected by electroejac- 32
20 Egg yolk
21 Sucrose
ulation. The sperm evaluation was carried out at 0, 2 and 4 h through the incubation time at 37 °C for the 33
22 Glycerol experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality 34
23 Ram semen throughout the incubation time always resulted lower (P 6 0.05) for the free-egg yolk diluents in relation 35
24 to BiladylÒ (control), obtaining the lowest values of sperm quality with the highest concentrations of 36
sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose 37
at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with BiladylÒ (at different sperm concen- 38
trations). The vitrification decreased drastically (P 6 0.05) the sperm quality when combinations of 39
sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with BiladylÒ at the lowest 40
sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the 41
sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on com- 42
binations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were 43
obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are 44
necessary to improve this technique and the use of diluents without egg yolk. 45
Ó 2015 Published by Elsevier Inc. 46
47

48
49
50 Introduction permeate the cell membrane has been routinely used to increase 62
membrane fluidity and partially dehydrating the cell, lowering 63
51 The conventional cryopreservation of spermatozoa is used in the freezing point, and thus reducing the number and size of intra- 64
52 small ruminants as part of breeding or conservation programs. It cellular ice crystals formed. Nevertheless, these cryoprotectants 65
53 is known that this technique, which involves cooling to 5 °C, equi- themselves can have a toxic effect on spermatozoa being this effect 66
54 libration at this temperature and freezing using vapors of liquid related to the concentration used and the time of cell exposure 67
55 nitrogen, causes important chemical–physical damage to the intra- [39]. In addition, egg yolk has been usually included as 68
56 cellular structures originated by changes in the osmotic balance non-permeable cryoprotector into sperm freezing extenders. The 69
57 and temperatures during the different steps. Thus, ice crystals main disadvantage is that egg yolk is a non-defined substance 70
58 are formed and as consequence, sperm membranes are affected, which leads to variability between batches. Moreover, it has an 71
59 an increase of lipid peroxidation is produced, sperm motility rates animal origin and it could be a way to transmit diseases. 72
60 and mitochondrial activity are decreased and processes associated During the last years, several studies have been carried out in 73
61 with cell death are induced [31]. Cryoprotective agents which order to avoid the use of cryoprotectans with animal origin. 74
Thus, extenders based on soybean or extenders containing sugars 75
with high molecular weight combined with permeable cryoprotec- 76
⇑ Corresponding author. Fax: +34 926 276 059.
tants such as glycerol has been studied for conventional sperm 77
E-mail address: pijimenez@jccm.es (P. Jiménez-Rabadán).

http://dx.doi.org/10.1016/j.cryobiol.2015.05.004
0011-2240/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: P. Jiménez-Rabadán et al., Effects of vitrification on ram spermatozoa using free-egg yolk extenders, Cryobiology (2015),
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2 P. Jiménez-Rabadán et al. / Cryobiology xxx (2015) xxx–xxx

78 freezing in different species [2,6,16–18,27,34]. Moreover, some BiladylÒ extender at concentrations of 20  106 and 140
79 investigations have been aimed to evaluate the effect of using per- 200  106 spermatozoa/mL (B5 and B50, respectively). 141
80 meable cryoprotectants [36], sucrose [37], or even a
81 cryoprotectants-free technique joined to other preservation meth- Semen collection 142
82 ods such as vitrification [11,13,36].
83 The vitrification process has been proposed as a novel technique Animal handling was performed in accordance with Spanish 143
84 widely used for embryo storage, but it has not been applied to rou- Animal Protection Regulation, RD 53/2013, which conforms to 144
85 tine sperm cryopreservation due to deleterious osmotic effect of European Union Regulation 2010/63. 145
86 high concentration of permeable cryoprotectans. This technique Semen was collected by electroejaculation from three 146
87 is based on the ultra-rapid freezing of the cell by direct immersion Manchega breed rams. The electroejaculation procedure was car- 147
88 in liquid nitrogen. The main advantage of this method is to prevent ried out using the protocol described by García-Álvarez et al. [9]. 148
89 the formation of ice crystals and the possibility to use extenders Males were anesthetized with xylazine (0.2 mg/kg RompunÒ 2% 149
90 free-egg yolk. Also, the procedure is faster, simpler in the applica- i.m.; Bayer S.A., Barcelona, Spain), the rectum was cleaned of faeces 150
91 tion and, more cost effective than the conventional cryopreserva- and the prepucial area was shaved and washed with physiologic 151
92 tion. As disadvantage, vitrification needs a high concentration of saline serum. A three electrode probe connected to a power source 152
93 cryoprotectant agents, and sperm cells are very sensitive to these that allowed voltage and amperage control was used (P.T. 153
94 agents [10]. Another limitation of this methodology is that so far, Electronics, Boring, OR, USA). Probe diameter, probe length and 154
95 vitrification has been performed using small volumes and open electrode length were 3.2, 35.0 and 6.6 cm, respectively. The EE 155
96 systems which not prevent direct contact with the liquid nitrogen regime consisted of consecutive series of 5-s pulses of similar volt- 156
97 [10,31]. Nevertheless, in recent studies in human, the spermatozoa age, each separated by 5-s break. Each series consisted of a total of 157
98 have been vitrified in large volume in straws obtaining good results four pulses. The initial voltage was 1V which was increased in each 158
99 [15,38]. series until a maximum of 5 V. Urine contamination was tested and 159
100 Taking into account the lack of knowledge about optimum ejaculates with urine were rejected. Semen from three males was 160
101 extenders to vitrify ram spermatozoa and about the impact of pooled in order to avoid individual variability and the pool was 161
102 sperm vitrification in this species, the present study was designed used to perform the different experiments. Experiments 1 and 2 162
103 to investigate: (1) the toxicity of sucrose and glycerol based on dif- were replicated 3 times. 163
104 ferent concentrations (sucrose at 0.03 M, 0.05 M, 0.015 M and
105 0.25 M; and glycerol at 3%, 7%, 14% and 18%) under incubation con- Semen evaluation 164
106 ditions; and (2) the effect of the vitrification using the best combi-
107 nation of the previous cryoprotectans. The effects of these factors For Experiment 1, sperm parameters were evaluated at 0, 2 and 165
108 were assessed in terms of sperm quality through the incubation 4 h of incubation at 37 °C after diluting with different extenders. 166
109 or at thawing, depending on objectives. The same sperm parameters were assessed after thawing in 167
Experiment 2. 168
Sperm motility was evaluated by Computer Assisted Semen 169
110 Materials and methods Analysis (CASA) using the Sperm Class Analyzer software (SCAÒ 170
2002, Microptic, Barcelona, Spain). Five lL of each diluted sperm 171
111 Experimental design sample were put on a pre-warmed Makler chamber at 37 °C. 172
Software settings were adjusted to ram spermatozoa and at least 173
112 Fig. 1 shows the experimental design followed in the present 200 spermatozoa were saved, recording the sperm motility as the 174
113 study, including the main experiments. percentage of motile spermatozoa (SM, %), defined as spermatozoa 175
114 Experiment 1 (Fig. 1A) was carried out to evaluate the effects with a curvilinear velocity (VCL) greater than 25 lm/s. 176
115 that different concentrations of sucrose and glycerol (sucrose: Aliquots of semen were used to conduct flow cytometry analy- 177
116 0.03, 0.05, 0.15 and 0.25 M; glycerol: 3%, 7%, 14% and 18%) added ses. Thus, it was assessed the membrane stability with YO-PRO-1, 178
117 to a SOF-based media have on sperm quality after 0, 2 and 4 h of the viability with PI, and the acrosome integrity by FICT-PNA. A 179
118 incubation at 37 °C. The values of sperm quality obtained from staining solution using TALP-HEPES was prepared by adding 180
119 the use of these extenders were compared with that obtained from 50 nM YO-PRO-1 (stock: 100 lM in DMSO) and 15 lM PI (stock: 181
120 the use of the conventional diluent in this breed, BiladylÒ (with 7.5 mM in milli-Q water). Twenty lL of sample were diluted in 182
121 20% of egg yolk and 7% glycerol). In the procedure of vitrification, 0.5 mL of staining solution in polypropylene tubes for flow cytom- 183
122 the sperm concentration used is usually very low and for this rea- etry and were kept in dark for 15 min. Acrosomal status was 184
123 son all media were used to low concentration assessed in a 12 lL PI and FITC-PNA 1 lg/mL staining solution. 185
124 (20  106 spermatozoa/mL). In addition, to discriminate a possible After incubation times, sperm samples were analyzed using a 186
125 negative effect of the dilution, another control with BiladylÒ was Cytomics FC500 flow cytometer (Beckman coulter, Inc. USA). A 187
126 performed to 200  106 spermatozoa/mL (B50). Moreover, a third 488 nm argon ion laser of the cytometer was used to excite the flu- 188
127 extender only based on SOF with BSA was tested since sucrose orochromes, namely YO-PRO-1, PI and FICT-PNA. Forward-scatter 189
128 and glycerol were added to a SOF-based extender. light (FSC) and side-scatter light (SSC) dot plots were used to dis- 190
129 Experiment 2 (Fig. 1B) was carried out to examine the behavior card debris. YO-PRO-1-/PI- spermatozoa were considered as intact 191
130 of ram spermatozoa subjected to a vitrification process using dif- spermatozoa (indicating live spermatozoa with intact plas- 192
131 ferent extenders with and without egg yolk, and it was compared malemma) and PNA-/PI- as living cells with intact acrosomes. 193
132 to the conventional cryopreservation protocol used in this species. Chromatin stability was assessed by using the SCSA (Sperm 194
133 The vitrification was performed diluting the sperm sample at Chromatin Structure Assay) technique (SCSA Diagnostics, Inc., 195
134 20  106 spermatozoa/mL with the combinations of extenders that Brookings, SD, USA) [5]. This technique is based on the susceptibil- 196
135 best results yield in the Experiment 1 (SOF-0.03 M and SOF-0.05 M ity of the sperm DNA to acid induced denaturation in situ and the 197
136 of sucrose with 3% or 7% glycerol respectively) and with BiladylÒ metachromatic staining AO. This stain fluoresces green when com- 198
137 extender at concentrations of 20  106 and bined with double-stranded DNA, and red when combined with 199
138 200  106 spermatozoa/mL (B5 and B50, respectively). The con- single stranded DNA (denaturated). Thawed spermatozoa were 200
139 ventional cryopreservation procedure was carried out using the diluted with TNE buffer to 2  106 cells/mL. Samples were flash 201

Please cite this article in press as: P. Jiménez-Rabadán et al., Effects of vitrification on ram spermatozoa using free-egg yolk extenders, Cryobiology (2015),
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Fig. 1. Experimental design. A: Experiment 1. B: Experiment 2. B50: BiladylÒ at 200  106 spermatozoa/mL; B5: BiladylÒ at 20  106 spermatozoa/mL; SOF: SOF-based
extender without cryoprotectants added; 0.03 M-3%: SOF-based extender with 0.03 M sucrose and 3% glycerol; 0.03 M-7%: SOF-based extender with 0.03 M sucrose and 7%
glycerol; 0.05 M-3%: SOF-based extender with 0.05 M sucrose and 3% glycerol; 0.05 M-7%: SOF-based extender with 0.05 M sucrose and 7% glycerol.

202 frozen in LN2 and stored at 80 °C until analysis. We calculated the straws 4 cm above the surface of nitrogen liquid. The straws were 227
203 DNA fragmentation Index (DFI) for each spermatozoon as the ratio subsequently plunged into liquid nitrogen and stored. 228
204 of red fluorescence respect to total fluorescence (red + green). High For vitrification, different extenders were used: BiladylÒ at a 229
205 values of DFI indicate chromatin abnormalities. The % DFI was cal- final concentration of 200 or 20  106 spermatozoa/mL and combi- 230
206 culated as the percentage of spermatozoa with DFI >25. nations of sucrose and glycerol in a SOF-based media (final sperm 231
concentration 20  106 spermatozoa/mL) in the following way: 232
0.03 M sucrose-3% glycerol, 0.03 M sucrose-7% glycerol, 0.05 M 233
207 Semen preservation sucrose-3% glycerol and 0.05 M sucrose-7% glycerol. The SOF media 234
used in this study was that described by Takahashi and First [40] 235
208 Semen preservation was performed in Experiment 2 using two without amino acids and supplemented with HEPES and 1% BSA. 236
209 different protocols. Thus, it was tested the effect of vitrification The diluted semen (100 lL) with different extenders was loaded 237
210 (direct immersion into liquid nitrogen) and it was compared to into 0.25 mL straws and they were inserted into 0.5 mL straws 238
211 conventional cryopreservation protocol (cooling to 5 °C for 2 h which were heat-sealed and directly plunged into liquid nitrogen. 239
212 and equilibration at this temperature for 2 h; total cooling time
213 4 h).
214 Conventional cryopreservation was performed using the com- Statistical analyses 240
215 mercial extender BiladylÒ and two final sperm concentrations were
216 reached: (i) a conventional of 200  106 spermatozoa/mL, and (ii) a Data preparation and statistical analyses were conducted using 241
217 concentration of 20  106 spermatozoa/mL, similar to that used in the R statistical packages [32]. Previous to data analysis, assump- 242
218 the vitrification procedure. This type of cryopreservation was car- tion of normality was tested and if not satisfied log or arcsin trans- 243
219 ried out in two steps. Firstly, non-glycerolated fraction was added formation was used. In Experiment 1, the effects of sucrose and 244
220 at 30 °C and the diluted semen (400 or 40  106 spermatozoa/mL) glycerol concentrations at 3 times of incubation on sperm quality 245
221 was cooled to 5 °C for 2 h. Then, it was further diluted (v:v to a final were examined using a linear regression model. The concentra- 246
222 concentration of 200 or 20  106 spermatozoa/mL) with the frac- tions of sucrose and glycerol given the best quality on 247
223 tion containing glycerol (7%). The diluted sperm samples were then Experiment 1 were then used jointly in Experiment 2. Thus, a lin- 248
224 held at 5 °C for 2 h more before freezing (equilibration time) and ear regression analysis was performed to examine the effects of 249
225 after that the diluted semen was loaded into 0.25 mL plastic straws extenders’ composition on sperm quality at thawing. Differences 250
226 and frozen on over nitrogen vapors for 10 min, by placing the among extenders within each cryopreservation procedure were 251

Please cite this article in press as: P. Jiménez-Rabadán et al., Effects of vitrification on ram spermatozoa using free-egg yolk extenders, Cryobiology (2015),
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252 tested using a multiple comparisons analysis considering the Fig. 1 in Supplementary material). There were no differences 277
253 Bonferroni adjustment. In all analyses, a threshold value of (P P 0.05) in the conventional cryopreservation between freezing 278
254 P < 0.05 was established to denote statistical significance. at 20 or 200  106 spermatozoa/mL, although the sperm motility 279
was slightly above for cryopreserved samples with the highest con- 280
centration. The samples vitrified showed a low sperm quality 281
255 Results (P 6 0.05) when combinations of sucrose and glycerol were used, 282
although the samples vitrified with BiladylÒ at 283
256 Experiment 1: Effect of cytotoxicity of sucrose and glycerol at different 20  106 spermatozoa/mL showed acceptable values for the most 284
257 concentrations on sperm quality after incubation period at 37 °C of sperm parameters, except for motility. Thus, for this treatment 285

Ò
the values of live spermatozoa with intact plasma membrane 286
258 Samples extended in Biladyl showed higher sperm quality (YO-PRO-1-/PI-) and live spermatozoa with intact acrosome 287
259 (P 6 0.05) than the other treatments evaluated including the con- (PNA-/PI-) were higher (P 6 0.05) than for the rest of extenders 288
260 trol using only SOF (Fig. 2A and B; Tables 1 and 2 in (Table 1; Fig. 1 in Supplementary material). 289
261 Supplementary material). There was no effect of the dilution
262 (P P 0.05), showing B50 and B5 similar values of sperm quality.
263 Adding sucrose or glycerol had a significant negative effect
Discussion 290
264 (P 6 0.05) on sperm quality for all sperm parameters assessed,
265 except for DFI which was unaffected. Moreover, higher sucrose or
Up to date, sperm cryopreservation in small ruminants has been 291
266 glycerol concentrations provided a decrease in sperm quality
performed by the conventional technique which involves three 292
267 (P 6 0.05) (Fig. 2A and B; Tables 1 and 2 in Supplementary
phases: cooling to 5 °C, equilibration at this temperature for a vari- 293
268 material).
able time and freezing on liquid nitrogen vapors. For this, exten- 294
269 Throughout the incubation period (from 0 to 4 h), treatments
ders based on different concentrations of egg yolk and glycerol 295
270 except BiladylÒ controls showed a progressive decrease (P 6 0.05)
have been widely used. Conventional techniques have been well 296
271 of sperm quality, which was more noticeable for samples contain-
investigated and it has been shown that intra- or extra-cellular 297
272 ing higher concentrations of sucrose or glycerol (Fig. 1A and B;
ice crystal formation and osmotic changes may occur during a slow 298
273 Tables 1 and 2 in Supplementary material).
freezing which led to extensive cell shrinkage and sperm damage 299
[7,41]. As alternative, vitrification has been proposed as a proce- 300
274 Experiment 2: Effect of vitrification on sperm quality after thawing dure that avoids the formation of ice crystal which leads to some 301
advantages in relation to conventional sperm cryopreservation. 302
275 The sperm quality was higher for samples cryopreserved in a Moreover, vitrification can be performed using extenders without 303
276 way conventional in relation to vitrification procedure (Table 1; egg yolk or permeable cryoprotectants avoiding the negative 304

Fig. 2. Effect of sucrose and glycerol cytotoxicity on spermatozoa under incubation conditions. Data are means ± sem. B5: BiladylÒ at 5  106 spz/straw; B50: BiladylÒ at
50  106 spz/straw; SOF: SOF-based extender without cryoprotectants added; SM: motile spermatozoa; YO-PRO-1-/PI-: intact spermatozoa; PNA-/PI-: living cells with intact
acrosomes; DFI: DNA fragmentation index.

Please cite this article in press as: P. Jiménez-Rabadán et al., Effects of vitrification on ram spermatozoa using free-egg yolk extenders, Cryobiology (2015),
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Table 1
Effect of extender and seminal concentration on sperm quality after thawing in vitrified samples.

Conventional cryopreservation Vitrification

SM YO-PRO1-/PI- PNA-/PI- DFI SM YO-PRO1-/PI- PNA-/PI- DFI
B50 34.65 ± 9.53 20.14 ± 4.65 20.73 ± 4.00 5.38 ± 1.07 0.10 ± 0.05 1.93 ± 2.09b 1.76 ± 1.94b 4.11 ± 0.80
B5 16.60 ± 9.53 30.66 ± 4.65 31.34 ± 4.00 5.75 ± 1.07 0 ± 0.05 12.78 ± 2.09ª 10.09 ± 1.94ª 6.08 ± 0.80
0.03 M-3% 0 ± 0.73 1.29 ± 2.95b 1.15 ± 2.74b 5.40 ± 1.13
0.03 M-7% 0 ± 0.73 0.91 ± 2.95b 1.16 ± 2.74b 4.71 ± 1.13
0.05 M-3% 0 ± 0.73 0.77 ± 2.95b 0.37 ± 2.74b 4.64 ± 1.13
0.05 M-7% 0 ± 0.73 0.24 ± 2.95b 0.67 ± 2.74b 4.00 ± 1.13

Data are means ± standard error. B50: BiladylÒ at final concentration of 50  106 spermatozoa/straw; B5: BiladylÒ at final concentration of 5  106 spermatozoa/straw;
0.03 M-3%: SOF-sucrose 0.03 M-3% glycerol; 0.03 M-7%: SOF-sucrose 0.03 M-7% glycerol; 0.05 M-3%: SOF-sucrose 0.05 M-3% glycerol; 0.05 M-7%: SOF-sucrose 0.05 M-7%
glycerol; SM: percentage of motile spermatozoa; YO-PRO-1-/PI-: intact spermatozoa; PNA-/PI-: living cells with intact acrosome; DFI: DNA fragmentation index.
Different letters show differences between extenders within each freezing procedure (P < 0.05).

305 effects of these substances [11,13] and using proteins and sugars as for extenders with combinations of 0.03 M and 0.05 M of sucrose 355
306 non-permeable extracellular cryoprotectants [19]. with 3% and 7% of glycerol, respectively. Contrary to our results, 356
307 Focusing on animal sperm vitrification, few studies have the vitrification is carried out in human spermatozoa with good 357
308 achieved good results, so now efforts are being put in the design results [12]. Nawroth et al. [30], who vitrified human spermatozoa 358
309 of new protocols which take into account new technologies and using extenders with and without cryoprotectans, observed the 359
310 the use of different extenders as a way to increase the success of best results when samples were vitrified with 360
311 the methodology. And more specifically for sheep, there is little free-cryoprotectans extenders. Similarly, the removal of permeable 361
312 information available in relation to the use of alternative methods cryoprotectans from the vitrification media caused an improve- 362
313 to preserve semen. For that reason, this study was firstly aimed to ment on the cryosurvival of fish and dog spermatozoa [26,37], 363
314 evaluate the toxicity of different cryoprotectants used for vitrifica- although Agha-Rahimi et al. [1] did not found differences in the 364
315 tion in other species (sucrose and/or glycerol) based on its concen- sperm quality of vitrified human spermatozoa using extenders 365
316 tration under incubation conditions. Subsequently, it was studied with and without cryoprotectans. 366
317 the effect of vitrification on the sperm quality at thawing when a The success in the vitrification has been more limited for others 367
318 combination of extenders at the concentrations which provided species. Thus, in rabbit [36] and kangaroo [25] the sperm vitrifica- 368
319 better results in the previous experiment were used. tion led to very few motile and/or viable cells post-thawing. The 369
320 In the first experiment, there was no differences in sperm qual- differences between studies could be due to the species. Shape 370
321 ity between controls B50 and B5, being the latter highly diluted. and size of the sperm head and the flagellum are factors that define 371
322 Contrary to our study, it has been showed that high dilutions of the cryosensivity of the cell [4,33,35]. A small size of head has been 372
323 the sperm samples lead to damage of spermatozoa [22,24]. related with a higher cryoresistance in some species [4]. Thus, 373
324 However, many assisted reproductive techniques, as sex-sorting, higher cryoresistance of vitrified spermatozoa in human could be 374
325 are performed using samples highly diluted without lack of sperm due to the small size of the sperm head in relation with others spe- 375
326 quality adding protector substances as egg yolk or seminal plasma cies [8]. 376
327 [24]. In our study, the B5 control was used to mimic the conditions In the other hand, our results showed that vitrification offered 377
328 of vitrification, since the most of authors vitrify with low concen- best protection to sperm plasma and acrosomal membrane of sper- 378
329 tration and volume [1,3,26,37] and in this way to reject the matozoa in samples more diluted using egg yolk-glyerol based 379
330 hypothesis of a possible damage due to dilution. extender than in those samples vitrified with higher sperm con- 380
331 In relation to sucrose toxicity, our study showed how an centration. However, the sperm motility was strongly affected in 381
332 increase of this sugar produced a reduction in sperm quality both conditions of concentration. It has been broadly assumed that 382
333 through the incubation period. Similar trend was observed when the high dilution of the sperm samples lead to damage of sperma- 383
334 concentration of glycerol increased. Thus, samples with 0.03 and tozoa [22], although Lehay et al. [21] showed that the high 384
335 0.05 M sucrose and 3% and 7% glycerol showed higher percentage pre-freezing dilution at 20  106 spermatozoa/mL improved the 385
336 of motile spermatozoa, viable spermatozoa and spermatozoa with viability, acrosome integrity and total motility after thawing in 386
337 intact acrosome. Contrary, higher concentrations of sucrose relation to samples more concentrated. The most authors have 387
338 (0.15 M and 0.25 M) and glycerol (14% and 18%) resulted in lower used low sperm volume and concentration to vitrify human sper- 388
339 sperm quality. In human, canine and rabbit, 0.25 M concentrations matozoa with good results [3,26,37]. In addition, Maxwell and 389
340 of sucrose had not a toxic effect on spermatozoa [14,36,37]. It has Jonhson [24] showed that egg-yolk offers some protection to 390
341 been described that sugars have a protector effect against osmotic diluted spermatozoa. It is possible that the joint action of 391
342 changes decreasing the ice formation and stabilizing the solute egg-yolk and low sperm concentration used in the vitrification of 392
343 concentration inside of sperm cell during osmotic stress as well ram spermatozoa would lead to higher protection of the spermato- 393
344 as, have the property of stabilizate the cell membrane [19,20,29]. zoa. The lack of positive results in our study could be due not only 394
345 Moreover, a synergistic effect between sugars and permeable cry- to the technique of vitrification or extenders, but also other factors 395
346 oprotectans has been reported in ram [28]. Therefore, the use of that were not considered, as the thawing rate. Recently, Mansilla 396
347 sugars in extenders could result in a decrease of concentration of et al. [23] found an influence of thawing temperature and time 397
348 permeable cryoprotectants used, thus reducing their negative on sperm quality in vitrified samples, with better values for the 398
349 effects on spermatozoa during the vitrification. highest temperature and shortest warming time. In our study the 399
350 Based on these observations, in a second experiment we exam- straws were thawing at 37 °C for 30 s being this combination very 400
351 ined in a vitrification procedure the effects of using extenders similar to that that showed the worst results in the previous study, 401
352 based on combinations of sucrose and glycerol at those concentra- 38 °C for 10 s [23]. 402
353 tions which provided the best results in the previous experiment. Based on results observed through experiments in the present 403
354 Results showed a decrease in the sperm quality after vitrification study, we can conclude that extenders based on combinations of 404

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405 sucrose and glycerol are not useful to vitrify ram spermatozoa. [16] M. Jafaroghli, B. Khalili, A. Farshad, M.J. Zamiri, The effect of supplementation 474
of cryopreservation diluents with sugars on the post-thawing fertility of ram 475
406 However, the vitrification using extenders with egg yolk and per- 476
semen, Small Rumin. Res. 96 (2011) 58–63.
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Please cite this article in press as: P. Jiménez-Rabadán et al., Effects of vitrification on ram spermatozoa using free-egg yolk extenders, Cryobiology (2015),
http://dx.doi.org/10.1016/j.cryobiol.2015.05.004