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Effects of vitrification on ram spermatozoa using free-egg yolk extenders
Cryobiology xxx (2015) xxx–xxx
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Effects of vitrification on ram spermatozoa using free-egg yolk extenders q Pilar Jiménez-Rabadán a,⇑, Olga García-Álvarez b, Ana Vidal b, Alejandro Maroto-Morales b, María Iniesta-Cuerda b, Manuel Ramón a, Enrique del Olmo b, Rocío Fernández-Santos b, J. Julián Garde b, Ana Josefa Soler b a b
Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain SaBio (IREC) CSIC-UCLM-JCCM, 02071 Albacete, Spain
a r t i c l e
i n f o
Article history: Received 4 December 2014 Accepted 13 May 2015 Available online xxxx Keywords: Vitrification Egg yolk Sucrose Glycerol Ram semen
a b s t r a c t The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (BiladylÒ with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4 h through the incubation time at 37 °C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P 6 0.05) for the free-egg yolk diluents in relation to BiladylÒ (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with BiladylÒ (at different sperm concentrations). The vitrification decreased drastically (P 6 0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with BiladylÒ at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk. Ó 2015 Elsevier Inc. All rights reserved.
Introduction The conventional cryopreservation of spermatozoa is used in small ruminants as part of breeding or conservation programs. It is known that this technique, which involves cooling to 5 °C, equilibration at this temperature and freezing using vapors of liquid nitrogen, causes important chemical–physical damage to the intracellular structures originated by changes in the osmotic balance and temperatures during the different steps. Thus, ice crystals
q Statement of funding: This work has been funded by the Castilla-La Mancha University (Gl20152913). ⇑ Corresponding author. Fax: +34 926 276 059. E-mail address: [email protected] (P. Jiménez-Rabadán).
are formed and as consequence, sperm membranes are affected, an increase of lipid peroxidation is produced, sperm motility rates and mitochondrial activity are decreased and processes associated with cell death are induced [31]. Cryoprotective agents which permeate the cell membrane has been routinely used to increase membrane fluidity and partially dehydrating the cell, lowering the freezing point, and thus reducing the number and size of intracellular ice crystals formed. Nevertheless, these cryoprotectants themselves can have a toxic effect on spermatozoa being this effect related to the concentration used and the time of cell exposure [39]. In addition, egg yolk has been usually included as non-permeable cryoprotector into sperm freezing extenders. The main disadvantage is that egg yolk is a non-defined substance which leads to variability between batches. Moreover, it has an animal origin and it could be a way to transmit diseases.
http://dx.doi.org/10.1016/j.cryobiol.2015.05.004 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
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
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During the last years, several studies have been carried out in order to avoid the use of cryoprotectans with animal origin. Thus, extenders based on soybean or extenders containing sugars with high molecular weight combined with permeable cryoprotectants such as glycerol has been studied for conventional sperm freezing in different species [2,6,16–18,27,34]. Moreover, some investigations have been aimed to evaluate the effect of using permeable cryoprotectants [36], sucrose [37], or even a cryoprotectants-free technique joined to other preservation methods such as vitrification [11,13,36]. The vitrification process has been proposed as a novel technique widely used for embryo storage, but it has not been applied to routine sperm cryopreservation due to deleterious osmotic effect of high concentration of permeable cryoprotectans. This technique is based on the ultra-rapid freezing of the cell by direct immersion in liquid nitrogen. The main advantage of this method is to prevent the formation of ice crystals and the possibility to use free-egg yolk extenders. Also, the procedure is faster, simpler in the application and, more cost effective than the conventional cryopreservation. As disadvantage, vitrification needs a high concentration of cryoprotectant agents, and sperm cells are very sensitive to these agents [10]. Another limitation of this methodology is that so far, vitrification has been performed using small volumes and open systems which not prevent direct contact with the liquid nitrogen [10,31]. Nevertheless, in recent studies in human, the spermatozoa have been vitrified in large volume in straws obtaining good results [15,38]. Taking into account the lack of knowledge about optimum extenders to vitrify ram spermatozoa and about the impact of sperm vitrification in this species, the present study was designed to investigate: (1) the toxicity of sucrose and glycerol based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.015 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) under incubation conditions; and (2) the effect of the vitrification using the best combination of the previous cryoprotectans. The effects of these factors were assessed in terms of sperm quality through the incubation or at thawing, depending on objectives.
Materials and methods Experimental design Fig. 1 shows the experimental design followed in the present study, including the main experiments. Experiment 1 (Fig. 1A) was carried out to evaluate the effects that different concentrations of sucrose and glycerol (sucrose: 0.03, 0.05, 0.15 and 0.25 M; glycerol: 3%, 7%, 14% and 18%) added to a SOF-based media have on sperm quality after 0, 2 and 4 h of incubation at 37 °C. The values of sperm quality obtained from the use of these extenders were compared with that obtained from the use of the conventional diluent in this breed, BiladylÒ (with 20% of egg yolk and 7% glycerol). In the procedure of vitrification, the sperm concentration used is usually very low and for this reason all media were used to low concentration (20 106 spermatozoa/mL). In addition, to discriminate a possible negative effect of the dilution, another control with BiladylÒ was performed to 200 106 spermatozoa/mL (B50). Moreover, a third extender only based on SOF with BSA was tested since sucrose and glycerol were added to a SOF-based extender. Experiment 2 (Fig. 1B) was carried out to examine the behavior of ram spermatozoa subjected to a vitrification process using different extenders with and without egg yolk, and it was compared to the conventional cryopreservation protocol used in this species. The vitrification was performed diluting the sperm sample at 20 106 spermatozoa/mL with the combinations of extenders that
best results yield in the Experiment 1 (SOF-0.03 M and SOF-0.05 M of sucrose with 3% or 7% glycerol respectively) and with BiladylÒ extender at concentrations of 20 106 and 200 106 spermatozoa/mL (B5 and B50, respectively). The conventional cryopreservation procedure was carried out using the BiladylÒ extender at concentrations of 20 106 and 200 106 spermatozoa/mL (B5 and B50, respectively). Semen collection Animal handling was performed in accordance with Spanish Animal Protection Regulation, RD 53/2013, which conforms to European Union Regulation 2010/63. Semen was collected by electroejaculation from three Manchega breed rams. The electroejaculation procedure was carried out using the protocol described by García-Álvarez et al. [9]. Males were anesthetized with xylazine (0.2 mg/kg RompunÒ 2% i.m.; Bayer S.A., Barcelona, Spain), the rectum was cleaned of faeces and the prepucial area was shaved and washed with physiologic saline serum. A three electrode probe connected to a power source that allowed voltage and amperage control was used (P.T. Electronics, Boring, OR, USA). Probe diameter, probe length and electrode length were 3.2, 35.0 and 6.6 cm, respectively. The EE regime consisted of consecutive series of 5-s pulses of similar voltage, each separated by 5-s break. Each series consisted of a total of four pulses. The initial voltage was 1V which was increased in each series until a maximum of 5 V. Urine contamination was tested and ejaculates with urine were rejected. Semen from three males was pooled in order to avoid individual variability and the pool was used to perform the different experiments. Experiments 1 and 2 were replicated 3 times. Semen evaluation For Experiment 1, sperm parameters were evaluated at 0, 2 and 4 h of incubation at 37 °C after diluting with different extenders. The same sperm parameters were assessed after thawing in Experiment 2. Sperm motility was evaluated by Computer Assisted Semen Analysis (CASA) using the Sperm Class Analyzer software (SCAÒ 2002, Microptic, Barcelona, Spain). Five lL of each diluted sperm sample were put on a pre-warmed Makler chamber at 37 °C. Software settings were adjusted to ram spermatozoa and at least 200 spermatozoa were saved, recording the sperm motility as the percentage of motile spermatozoa (SM, %), defined as spermatozoa with a curvilinear velocity (VCL) greater than 25 lm/s. Aliquots of semen were used to conduct flow cytometry analyses. Thus, it was assessed the membrane stability with YO-PRO-1, the viability with PI, and the acrosome integrity by FICT-PNA. A staining solution using TALP-HEPES was prepared by adding 50 nM YO-PRO-1 (stock: 100 lM in DMSO) and 15 lM PI (stock: 7.5 mM in milli-Q water). Twenty lL of sample were diluted in 0.5 mL of staining solution in polypropylene tubes for flow cytometry and were kept in dark for 15 min. Acrosomal status was assessed in a 12 lL PI and FITC-PNA 1 lg/mL staining solution. After incubation times, sperm samples were analyzed using a Cytomics FC500 flow cytometer (Beckman coulter, Inc. USA). A 488 nm argon ion laser of the cytometer was used to excite the fluorochromes, namely YO-PRO-1, PI and FICT-PNA. Forward-scatter light (FSC) and side-scatter light (SSC) dot plots were used to discard debris. YO-PRO-1-/PI- spermatozoa were considered as intact spermatozoa (indicating live spermatozoa with intact plasmalemma) and PNA-/PI- as living cells with intact acrosomes. Chromatin stability was assessed by using the SCSA (Sperm Chromatin Structure Assay) technique (SCSA Diagnostics, Inc., Brookings, SD, USA) [5]. This technique is based on the
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
P. Jiménez-Rabadán et al. / Cryobiology xxx (2015) xxx–xxx
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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.
susceptibility of the sperm DNA to acid induced denaturation in situ and the metachromatic staining AO. This stain fluoresces green when combined with double-stranded DNA, and red when combined with single stranded DNA (denaturated). Thawed spermatozoa were diluted with TNE buffer to 2 106 cells/mL. Samples were flash frozen in LN2 and stored at 80 °C until analysis. We calculated the DNA fragmentation Index (DFI) for each spermatozoon as the ratio of red fluorescence respect to total fluorescence (red + green). High values of DFI indicate chromatin abnormalities. The % DFI was calculated as the percentage of spermatozoa with DFI >25.
Semen preservation Semen preservation was performed in Experiment 2 using two different protocols. Thus, it was tested the effect of vitrification (direct immersion into liquid nitrogen) and it was compared to conventional cryopreservation protocol (cooling to 5 °C for 2 h and equilibration at this temperature for 2 h; total cooling time 4 h). Conventional cryopreservation was performed using the commercial extender BiladylÒ and two final sperm concentrations were reached: (i) a conventional of 200 106 spermatozoa/mL, and (ii) a concentration of 20 106 spermatozoa/mL, similar to that used in the vitrification procedure. This type of cryopreservation was carried out in two steps. Firstly, non-glycerolated fraction was added at 30 °C and the diluted semen (400 or 40 106 spermatozoa/mL)
was cooled to 5 °C for 2 h. Then, it was further diluted (v:v to a final concentration of 200 or 20 106 spermatozoa/mL) with the fraction containing glycerol (7%). The diluted sperm samples were then held at 5 °C for 2 h more before freezing (equilibration time) and after that the diluted semen was loaded into 0.25 mL plastic straws and frozen on over nitrogen vapors for 10 min, by placing the straws 4 cm above the surface of nitrogen liquid. The straws were subsequently plunged into liquid nitrogen and stored. For vitrification, different extenders were used: BiladylÒ at a final concentration of 200 or 20 106 spermatozoa/mL and combinations of sucrose and glycerol in a SOF-based media (final sperm concentration 20 106 spermatozoa/mL) in the following way: 0.03 M sucrose-3% glycerol, 0.03 M sucrose-7% glycerol, 0.05 M sucrose-3% glycerol and 0.05 M sucrose-7% glycerol. The SOF media used in this study was that described by Takahashi and First [40] without amino acids and supplemented with HEPES and 1% BSA. The diluted semen (100 lL) with different extenders was loaded into 0.25 mL straws and they were inserted into 0.5 mL straws which were heat-sealed and directly plunged into liquid nitrogen.
Statistical analyses Data preparation and statistical analyses were conducted using the R statistical packages [32]. Previous to data analysis, assumption of normality was tested and if not satisfied log or arcsin transformation was used. In Experiment 1, the effects of sucrose and glycerol concentrations at 3 times of incubation on sperm quality
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
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were examined using a linear regression model. The concentrations of sucrose and glycerol given the best quality on Experiment 1 were then used jointly in Experiment 2. Thus, a linear regression analysis was performed to examine the effects of extenders’ composition on sperm quality at thawing. Differences among extenders within each cryopreservation procedure were tested using a multiple comparisons analysis considering the Bonferroni adjustment. In all analyses, a threshold value of P < 0.05 was established to denote statistical significance.
Results Experiment 1: Effect of cytotoxicity of sucrose and glycerol at different concentrations on sperm quality after incubation period at 37 °C Samples extended in BiladylÒ showed higher sperm quality (P 6 0.05) than the other treatments evaluated including the control using only SOF (Fig. 2A and B; Tables 1 and 2 in Supplementary material). There was no effect of the dilution (P > 0.05), showing B50 and B5 similar values of sperm quality. Adding sucrose or glycerol had a significant negative effect (P 6 0.05) on sperm quality for all sperm parameters assessed, except for DFI which was unaffected. Moreover, higher sucrose or glycerol concentrations provided a decrease in sperm quality (P 6 0.05) (Fig. 2A and B; Tables 1 and 2 in Supplementary material). Throughout the incubation period (from 0 to 4 h), treatments except BiladylÒ controls showed a progressive decrease (P 6 0.05) of sperm quality, which was more noticeable for samples containing higher concentrations of sucrose or glycerol (Fig. 2A and B; Tables 1 and 2 in Supplementary material).
Experiment 2: Effect of vitrification on sperm quality after thawing The sperm quality was higher for samples cryopreserved in a way conventional in relation to vitrification procedure (Table 1; Fig. 1 in Supplementary material). There were no differences (P P 0.05) in the conventional cryopreservation between freezing at 20 or 200 106 spermatozoa/mL, although the sperm motility was slightly above for cryopreserved samples with the highest concentration. The samples vitrified showed a low sperm quality (P 6 0.05) when combinations of sucrose and glycerol were used, although the samples vitrified with BiladylÒ at 20 106 spermatozoa/mL showed acceptable values for the most of sperm parameters, except for motility. Thus, for this treatment the values of live spermatozoa with intact plasma membrane (YO-PRO-1-/PI-) and live spermatozoa with intact acrosome (PNA-/PI-) were higher (P 6 0.05) than for the rest of extenders (Table 1; Fig. 1 in Supplementary material).
Discussion Up to date, sperm cryopreservation in small ruminants has been performed by the conventional technique which involves three phases: cooling to 5 °C, equilibration at this temperature for a variable time and freezing on liquid nitrogen vapors. For this, extenders based on different concentrations of egg yolk and glycerol have been widely used. Conventional techniques have been well investigated and it has been shown that intra- or extra-cellular ice crystal formation and osmotic changes may occur during a slow freezing which led to extensive cell shrinkage and sperm damage [7,41]. As alternative, vitrification has been proposed as a
Fig. 2. Effect of sucrose and glycerol cytotoxicity on spermatozoa under incubation conditions. Data are means ± sem. B5: BiladylÒ at 20 106 spz/mL; B50: BiladylÒ at 200 106 spz/mL; 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), http://dx.doi.org/10.1016/j.cryobiol.2015.05.004
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P. Jiménez-Rabadán et al. / Cryobiology xxx (2015) xxx–xxx Table 1 Effect of extender and sperm concentration on seminal quality after thawing in vitrified and conventionally cryopreserved samples. Conventional cryopreservation
B50 B5 0.03 M-3% 0.03 M-7% 0.05 M-3% 0.05 M-7%
Vitrification
SM
YO-PRO1-/PI-
PNA-/PI-
DFI
SM
YO-PRO1-/PI-
PNA-/PI-
DFI
34.65 ± 9.53 16.60 ± 9.53
20.14 ± 4.65 30.66 ± 4.65
20.73 ± 4.00 31.34 ± 4.00
5.38 ± 1.07 5.75 ± 1.07
0.10 ± 0.05 0 ± 0.05 0 ± 0.73 0 ± 0.73 0 ± 0.73 0 ± 0.73
1.93 ± 2.09b 12.78 ± 2.09ª 1.29 ± 2.95b 0.91 ± 2.95b 0.77 ± 2.95b 0.24 ± 2.95b
1.76 ± 1.94b 10.09 ± 1.94ª 1.15 ± 2.74b 1.16 ± 2.74b 0.37 ± 2.74b 0.67 ± 2.74b
4.11 ± 0.80 6.08 ± 0.80 5.40 ± 1.13 4.71 ± 1.13 4.64 ± 1.13 4.00 ± 1.13
Data are means ± standard error. B50: BiladylÒ at final concentration of 200 106 spermatozoa/mL; B5: BiladylÒ at final concentration of 20 106 spermatozoa/mL; 0.03 M3%: 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).
procedure that avoids the formation of ice crystal which leads to some advantages in relation to conventional sperm cryopreservation. Moreover, vitrification can be performed using extenders without egg yolk or permeable cryoprotectants avoiding the negative effects of these substances [11,13] and using proteins and sugars as non-permeable extracellular cryoprotectants [19]. Focusing on animal sperm vitrification, few studies have achieved good results, so now efforts are being put in the design of new protocols which take into account new technologies and the use of different extenders as a way to increase the success of the methodology. And more specifically for sheep, there is little information available in relation to the use of alternative methods to preserve semen. For that reason, this study was firstly aimed to evaluate the toxicity of different cryoprotectants used for vitrification in other species (sucrose and/or glycerol) based on its concentration under incubation conditions. Subsequently, it was studied the effect of vitrification on the sperm quality at thawing when a combination of extenders at the concentrations which provided better results in the previous experiment were used. In the first experiment, there was no differences in sperm quality between controls B50 and B5, being the latter highly diluted. Contrary to our study, it has been showed that high dilutions of the sperm samples lead to damage of spermatozoa [22,24]. However, many assisted reproductive techniques, as sex-sorting, are performed using samples highly diluted without lack of sperm quality adding protector substances as egg yolk or seminal plasma [24]. In our study, the B5 control was used to mimic the conditions of vitrification, since the most of authors vitrify with low concentration and volume [1,3,26,37] and in this way to reject the hypothesis of a possible damage due to dilution. In relation to sucrose toxicity, our study showed how an increase of this sugar produced a reduction in sperm quality through the incubation period. Similar trend was observed when concentration of glycerol increased. Thus, samples with 0.03 and 0.05 M sucrose and 3% and 7% glycerol showed higher percentage of motile spermatozoa, viable spermatozoa and spermatozoa with intact acrosome. Contrary, higher concentrations of sucrose (0.15 M and 0.25 M) and glycerol (14% and 18%) resulted in lower sperm quality. In human, canine and rabbit, 0.25 M concentrations of sucrose had not a toxic effect on spermatozoa [14,36,37]. It has been described that sugars have a protector effect against osmotic changes decreasing the ice formation and stabilizing the solute concentration inside of sperm cell during osmotic stress as well as, have the property of stabilizate the cell membrane [19,20,29]. Moreover, a synergistic effect between sugars and permeable cryoprotectans has been reported in ram [28]. Therefore, the use of sugars in extenders could result in a decrease of concentration of permeable cryoprotectants used, thus reducing their negative effects on spermatozoa during the vitrification. Based on these observations, in a second experiment we examined in a vitrification procedure the effects of using extenders
based on combinations of sucrose and glycerol at those concentrations which provided the best results in the previous experiment. Results showed a decrease in the sperm quality after vitrification for extenders with combinations of 0.03 M and 0.05 M of sucrose with 3% and 7% of glycerol, respectively. Contrary to our results, the vitrification was carried out in human spermatozoa with good results [12]. Nawroth et al. [30], who vitrified human spermatozoa using extenders with and without cryoprotectans, observed the best results when samples were vitrified with free-cryoprotectans extenders. Similarly, the removal of permeable cryoprotectans from the vitrification media caused an improvement on the cryosurvival of fish and dog spermatozoa [26,37], although Agha-Rahimi et al. [1] did not found differences in the sperm quality of vitrified human spermatozoa using extenders with and without cryoprotectans. The success in the vitrification has been more limited for others species. Thus, in rabbit [36] and kangaroo [25] the sperm vitrification led to very few motile and/or viable cells post-thawing. The differences between studies could be due to the species. Shape and size of the sperm head and the flagellum are factors that define the cryosensivity of the cell [4,33,35]. A small size of head has been related with a higher cryoresistance in some species [4]. Thus, higher cryoresistance of vitrified spermatozoa in human could be due to the small size of the sperm head in relation with others species [8]. In the other hand, our results showed that vitrification offered best protection to sperm plasma and acrosomal membrane of spermatozoa in samples more diluted using egg yolk-glyerol based extender than in those samples vitrified with higher sperm concentration. However, the sperm motility was strongly affected in both conditions of concentration. It has been broadly assumed that the high dilution of the sperm samples lead to damage of spermatozoa [22], although Lehay et al. [21] showed that the high pre-freezing dilution at 20 106 spermatozoa/mL improved the viability, acrosome integrity and total motility after thawing in relation to samples more concentrated. The most authors have used low sperm volume and concentration to vitrify human spermatozoa with good results [3,26,37]. In addition, Maxwell and Jonhson [24] showed that egg yolk offers some protection to diluted spermatozoa. It is possible that the joint action of egg yolk and low sperm concentration used in the vitrification of ram spermatozoa would lead to higher protection of the spermatozoa. The lack of positive results in our study could be due not only to the technique of vitrification or extenders, but also other factors that were not considered, as the thawing rate. Recently, Mansilla et al. [23] found an influence of thawing temperature and time on sperm quality in vitrified samples, with better values for the highest temperature and shortest warming time. In our study the straws were thawed at 37 °C for 30 s being this combination very similar to that that showed the worst results in the previous study, 38 °C for 10 s [23].
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
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Based on results observed through experiments in the present study, we can conclude that extenders based on combinations of sucrose and glycerol are not useful to vitrify ram spermatozoa. However, the vitrification using extenders with egg yolk and performed it to low sperm concentration offers a possibility for the future use of this technique. More studies are needed to improve both the vitrification procedure and extenders. Acknowledgments We want to acknowledge Jose María Luján for his help during sperm samples collection. M Ramón is supported by the DOC-INIA program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cryobiol.2015.05. 004. References [1] A. Agha-Rahimi, M.A. Khalili, A. Nabi, S. Ashourzadeh, Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding, Reprod. Biomed. Online 28 (2014) 352–358. [2] V.A. Aires, K.D. Hinsch, F. Mueller-Schloesser, K. Bogner, S. Mueller-Schloesser, E. Hinsch, In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen, Theriogenology 60 (2003) 269–279. [3] Y. Chen, L. Li, Y. Qian, C. Xu, Y. Zhu, H. Huang, F. Jin, Y. Ye, Small-volume vitrification for human spermatozoa in the absence of cryoprotectans by using Cryotop, Andrologia (2014), http://dx.doi.org/10.1111/and.12320. [4] M.C. Esteso, A.J. Soler, M.R. Fernández-Santos, A.A. Quintero-Moreno, J.J. Garde, Functional significance of the sperm head morphometric size and shape for determining freezability in Iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples, J. Androl. 27 (2006) 662–670. [5] D. Evenson, L. Jost, Sperm chromatin structure assay is useful for fertility assessment, Methods Cell Sci. 22 (2000) 169–189. [6] M. Forouzanfar, M. Sharafi, S.M. Hosseini, S. Ostadhosseini, M. Hajian, L. Hossein, P. Abedi, N. Nili, H.R. Rahmani, M.H. Nasr-Esfahani, In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen, Theriogenology 73 (2010) 480–487. [7] D. Gao, J. Liu, C. Liu, L.E. Mcgann, P.F. Watson, F.W. Kleinhans, P. Mazur, E.S. Critser, J.K. Critser, Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol, Hum. Reprod. 10 (1995) 1109–1122. [8] D. Gao, P. Mazur, J. Critser, Fundamental cryobiology of mammalian spermatozoa, in: A.M. Karow, J.K. Critser (Eds.), Reproductive Tissue Banking, Academic Press, London, 1997. [9] O. García-Álvarez, A. Maroto-Morales, F. Martínez-Pastor, J.J. Garde, M. Ramón, M.R. Fernández-Santos, M.C. Esteso, M.D. Pérez-Guzmán, A.J. Soler, Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: electroejaculation and post-mortem collection, Theriogenology 72 (2009) 160–168. [10] E. Isachenko, V. Isachenko, I.I. Katkov, S. Dessole, F. Nawroth, Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success, Reprod. Biomed. Online 6 (2003) 191–200. [11] V. Isachenko, E. Isachenko, I.I. Katkov, M. Montag, S. Dessole, F. Nawroth, H. Van der Ven, Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability, Biol. Reprod. 71 (2004) 1167–1173. [12] E. Isachenko, V. Isachencko, I.I. Katkov, G. Rahimi, T. Schöndorf, P. Mallmann, S. Dessole, F. Nawrtoh, DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification, Hum. Reprod. 19 (2004) 932–939. [13] V. Isachenko, E. Isachenko, A.M. Petrunkina, R. Sanchez, Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies, Reprod. Fertil. Dev. 24 (2012) 323–326. [14] E. Isachenko, V. Isachenko, J.M. Weiss, R. Kreienberg, I.I. Katkov, M. Schulz, A.G. Lulat, M.J. Risopatron, R. Sánchez, Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose, Reproduction 136 (2008) 167– 173. [15] E. Isachencko, P. Mallamann, G. Rahimi, J. Risopatron, M. Schulz, V. Isachenko, R. Sánchez, Vitrification technique-new possibilities for male gamete lowtemperature storage, in: I. Katkov (Ed.), Current Frontiers in Cryobiology, Croatia, 2012, pp. 44–76.
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Mazur, Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose, Cryobiology 41 (2000) 204–231. [20] C.H. Koshimoto, P. Mazur, Effects of cooling and warming rate to and from 70 °C, and effect of further cooling from 70 to 196 °C on the motility of mouse spermatozoa, Biol. Reprod. 66 (2002) 1477–1484. [21] T. Lehay, J.I. Marti, N. Mendoza, P. Pérez-Pé, T. Muiño-Blanco, J.A. CebriánPérez, G. Evans, W.M.C. Maxwell, High pre-freezing dilution improves postthaw function of ram spermatozoa, Anim. Reprod. Sci. 119 (2010) 137–146. [22] T. Mann, The Biochemistry of Semen and of the Male Reproductive, Tract. Methuen and Co., Ltd., London, 1964. [23] M.A. Mansilla, O. Merino, J. Risopatrón, V. Isachenko, E. Isachenko, R. Sánchez, High temperature is essential for preserved human sperm function during the devitrification process, Andrologia (2014), http://dx.doi.org/10.1111/ and.12406. [24] W.M.C. Maxwell, L.A. Johnson, Physiology of spermatozoa at high dilution rates: the influence of seminal plasma, Theriogenology 52 (1999) 1353–1362. [25] R. McClean, W.V. Holt, Y.P. Zee, A. Lisle, S.D. Johnston, The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function, Cryobiology 57 (2008) 297–303. [26] O. Merino, J. Risopatrón, R. Sánchez, E. Isachenko, E. Figueroa, I. Valdebenito, V. Isachenko, Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness, Anim. Reprod. Sci. 124 (2011) 125–131. [27] F.C. Molinia, G. Evans, P.I. Quintana Casares, W.C.M. Maxwell, Effect of monosaccharides and disaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa, Anim. Reprod. Sci. 36 (1994) 113–122. [28] A. Najafi, M. Zhandi, A. Towhidi, M. Shafari, A.A. Sharif, M.K. Motlagh, F. Martínez-Pastor, Trehalose and glycerol have a dose-dependent synergistic effect on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender, Cryobiology 66 (2013) 275–282. [29] N. Nakagata, T. Takeshim, High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation, Theriogenology 37 (1992) 1263–1291. [30] F. Nawrtoh, V. Isachenko, S. Dessole, G. Rahimi, M. Farina, N. Vargiu, P. Mallmann, M. Dattena, G. Capobianco, D. Peters, I. Orth, E. Isachenko, Vitrification of human spermatozoa without cryoprotectans, CryoLetters 23 (2002) 93–102. [31] M. O’Conell, N. McClure, E.M. Lewis, The effects of cryopreservation on sperm morphology, motility and mitochondrial function, Hum. Reprod. 17 (2002) 704–709. doi: 10/2016/j.theriogenology.2014.09.007. [32] R Core Team, A Language and Environmental for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2013. [33] M. Ramón, M.D. Pérez-Guzmán, P. Jiménez-Rabadán, M.C. Esteso, O. GarcíaÁlvarez, A. Maroto-Morales, L. Anel-López, A.J. Soler, M.R. Fernández-Santos, J.J. Garde, Sperm cell population dynamics in ram semen during the cryopreservation process, PLoS One 8 (2013) e59189. [34] D.J. Roof, S. Bowley, L.L. Price, D.J. Matsas, Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing, Theriogenology 77 (2012) 412–420. [35] J.L. Ros-Santaella, A.E. Dominguez-Rebolledo, J.J. Garde, Sperm flagellum volumen determines freezability in red deer spermatozoa, PLoS One 9 (2014) e112382. [36] M.P. Rosato, N. Iaffaldano, Cryopreservation of rabbit semen: Comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification, Theriogenology 79 (2013) 508–516. [37] R. Sánchez, J. Risopatrón, M. Schulz, J. Villegas, V. Isachenko, R. Kreingberg, E. Isachenko, Canine sperm vitrification with sucrose: effect of sperm, Andrologia 43 (2011) 233–241. [38] M. Slabbert, S.S. du Plessis, C. Huyser, Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa, Andrologia (2014), http://dx.doi.org/10.1111/and.12307. [39] J.E. Swain, G.D. Smith, Cryoprotectants, in: R.C. Chian, P. Quinn (Eds.), Fertility Cryopreservation, Cambridge University Press, New York, 2010, pp. 24–38. [40] Y. Takahashi, N.L. First, In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids, and vitamins, Theriogenology 37 (1992) 963–968. [41] P.F. Watson, Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function, Reprod. Fertil. Dev. 7 (1995) 871–891.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Effects of vitrification on ram spermatozoa using free-egg yolk extenders q Pilar Jiménez-Rabadán a,⇑, Olga García-Álvarez b, Ana Vidal b, Alejandro Maroto-Morales b, María Iniesta-Cuerda b, Manuel Ramón a, Enrique del Olmo b, Rocío Fernández-Santos b, J. Julián Garde b, Ana Josefa Soler b a b
Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain SaBio (IREC) CSIC-UCLM-JCCM, 02071 Albacete, Spain
a r t i c l e
i n f o
Article history: Received 4 December 2014 Accepted 13 May 2015 Available online xxxx Keywords: Vitrification Egg yolk Sucrose Glycerol Ram semen
a b s t r a c t The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (BiladylÒ with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4 h through the incubation time at 37 °C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P 6 0.05) for the free-egg yolk diluents in relation to BiladylÒ (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with BiladylÒ (at different sperm concentrations). The vitrification decreased drastically (P 6 0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with BiladylÒ at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk. Ó 2015 Elsevier Inc. All rights reserved.
Introduction The conventional cryopreservation of spermatozoa is used in small ruminants as part of breeding or conservation programs. It is known that this technique, which involves cooling to 5 °C, equilibration at this temperature and freezing using vapors of liquid nitrogen, causes important chemical–physical damage to the intracellular structures originated by changes in the osmotic balance and temperatures during the different steps. Thus, ice crystals
q Statement of funding: This work has been funded by the Castilla-La Mancha University (Gl20152913). ⇑ Corresponding author. Fax: +34 926 276 059. E-mail address: [email protected] (P. Jiménez-Rabadán).
are formed and as consequence, sperm membranes are affected, an increase of lipid peroxidation is produced, sperm motility rates and mitochondrial activity are decreased and processes associated with cell death are induced [31]. Cryoprotective agents which permeate the cell membrane has been routinely used to increase membrane fluidity and partially dehydrating the cell, lowering the freezing point, and thus reducing the number and size of intracellular ice crystals formed. Nevertheless, these cryoprotectants themselves can have a toxic effect on spermatozoa being this effect related to the concentration used and the time of cell exposure [39]. In addition, egg yolk has been usually included as non-permeable cryoprotector into sperm freezing extenders. The main disadvantage is that egg yolk is a non-defined substance which leads to variability between batches. Moreover, it has an animal origin and it could be a way to transmit diseases.
http://dx.doi.org/10.1016/j.cryobiol.2015.05.004 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
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
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During the last years, several studies have been carried out in order to avoid the use of cryoprotectans with animal origin. Thus, extenders based on soybean or extenders containing sugars with high molecular weight combined with permeable cryoprotectants such as glycerol has been studied for conventional sperm freezing in different species [2,6,16–18,27,34]. Moreover, some investigations have been aimed to evaluate the effect of using permeable cryoprotectants [36], sucrose [37], or even a cryoprotectants-free technique joined to other preservation methods such as vitrification [11,13,36]. The vitrification process has been proposed as a novel technique widely used for embryo storage, but it has not been applied to routine sperm cryopreservation due to deleterious osmotic effect of high concentration of permeable cryoprotectans. This technique is based on the ultra-rapid freezing of the cell by direct immersion in liquid nitrogen. The main advantage of this method is to prevent the formation of ice crystals and the possibility to use free-egg yolk extenders. Also, the procedure is faster, simpler in the application and, more cost effective than the conventional cryopreservation. As disadvantage, vitrification needs a high concentration of cryoprotectant agents, and sperm cells are very sensitive to these agents [10]. Another limitation of this methodology is that so far, vitrification has been performed using small volumes and open systems which not prevent direct contact with the liquid nitrogen [10,31]. Nevertheless, in recent studies in human, the spermatozoa have been vitrified in large volume in straws obtaining good results [15,38]. Taking into account the lack of knowledge about optimum extenders to vitrify ram spermatozoa and about the impact of sperm vitrification in this species, the present study was designed to investigate: (1) the toxicity of sucrose and glycerol based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.015 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) under incubation conditions; and (2) the effect of the vitrification using the best combination of the previous cryoprotectans. The effects of these factors were assessed in terms of sperm quality through the incubation or at thawing, depending on objectives.
Materials and methods Experimental design Fig. 1 shows the experimental design followed in the present study, including the main experiments. Experiment 1 (Fig. 1A) was carried out to evaluate the effects that different concentrations of sucrose and glycerol (sucrose: 0.03, 0.05, 0.15 and 0.25 M; glycerol: 3%, 7%, 14% and 18%) added to a SOF-based media have on sperm quality after 0, 2 and 4 h of incubation at 37 °C. The values of sperm quality obtained from the use of these extenders were compared with that obtained from the use of the conventional diluent in this breed, BiladylÒ (with 20% of egg yolk and 7% glycerol). In the procedure of vitrification, the sperm concentration used is usually very low and for this reason all media were used to low concentration (20 106 spermatozoa/mL). In addition, to discriminate a possible negative effect of the dilution, another control with BiladylÒ was performed to 200 106 spermatozoa/mL (B50). Moreover, a third extender only based on SOF with BSA was tested since sucrose and glycerol were added to a SOF-based extender. Experiment 2 (Fig. 1B) was carried out to examine the behavior of ram spermatozoa subjected to a vitrification process using different extenders with and without egg yolk, and it was compared to the conventional cryopreservation protocol used in this species. The vitrification was performed diluting the sperm sample at 20 106 spermatozoa/mL with the combinations of extenders that
best results yield in the Experiment 1 (SOF-0.03 M and SOF-0.05 M of sucrose with 3% or 7% glycerol respectively) and with BiladylÒ extender at concentrations of 20 106 and 200 106 spermatozoa/mL (B5 and B50, respectively). The conventional cryopreservation procedure was carried out using the BiladylÒ extender at concentrations of 20 106 and 200 106 spermatozoa/mL (B5 and B50, respectively). Semen collection Animal handling was performed in accordance with Spanish Animal Protection Regulation, RD 53/2013, which conforms to European Union Regulation 2010/63. Semen was collected by electroejaculation from three Manchega breed rams. The electroejaculation procedure was carried out using the protocol described by García-Álvarez et al. [9]. Males were anesthetized with xylazine (0.2 mg/kg RompunÒ 2% i.m.; Bayer S.A., Barcelona, Spain), the rectum was cleaned of faeces and the prepucial area was shaved and washed with physiologic saline serum. A three electrode probe connected to a power source that allowed voltage and amperage control was used (P.T. Electronics, Boring, OR, USA). Probe diameter, probe length and electrode length were 3.2, 35.0 and 6.6 cm, respectively. The EE regime consisted of consecutive series of 5-s pulses of similar voltage, each separated by 5-s break. Each series consisted of a total of four pulses. The initial voltage was 1V which was increased in each series until a maximum of 5 V. Urine contamination was tested and ejaculates with urine were rejected. Semen from three males was pooled in order to avoid individual variability and the pool was used to perform the different experiments. Experiments 1 and 2 were replicated 3 times. Semen evaluation For Experiment 1, sperm parameters were evaluated at 0, 2 and 4 h of incubation at 37 °C after diluting with different extenders. The same sperm parameters were assessed after thawing in Experiment 2. Sperm motility was evaluated by Computer Assisted Semen Analysis (CASA) using the Sperm Class Analyzer software (SCAÒ 2002, Microptic, Barcelona, Spain). Five lL of each diluted sperm sample were put on a pre-warmed Makler chamber at 37 °C. Software settings were adjusted to ram spermatozoa and at least 200 spermatozoa were saved, recording the sperm motility as the percentage of motile spermatozoa (SM, %), defined as spermatozoa with a curvilinear velocity (VCL) greater than 25 lm/s. Aliquots of semen were used to conduct flow cytometry analyses. Thus, it was assessed the membrane stability with YO-PRO-1, the viability with PI, and the acrosome integrity by FICT-PNA. A staining solution using TALP-HEPES was prepared by adding 50 nM YO-PRO-1 (stock: 100 lM in DMSO) and 15 lM PI (stock: 7.5 mM in milli-Q water). Twenty lL of sample were diluted in 0.5 mL of staining solution in polypropylene tubes for flow cytometry and were kept in dark for 15 min. Acrosomal status was assessed in a 12 lL PI and FITC-PNA 1 lg/mL staining solution. After incubation times, sperm samples were analyzed using a Cytomics FC500 flow cytometer (Beckman coulter, Inc. USA). A 488 nm argon ion laser of the cytometer was used to excite the fluorochromes, namely YO-PRO-1, PI and FICT-PNA. Forward-scatter light (FSC) and side-scatter light (SSC) dot plots were used to discard debris. YO-PRO-1-/PI- spermatozoa were considered as intact spermatozoa (indicating live spermatozoa with intact plasmalemma) and PNA-/PI- as living cells with intact acrosomes. Chromatin stability was assessed by using the SCSA (Sperm Chromatin Structure Assay) technique (SCSA Diagnostics, Inc., Brookings, SD, USA) [5]. This technique is based on the
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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.
susceptibility of the sperm DNA to acid induced denaturation in situ and the metachromatic staining AO. This stain fluoresces green when combined with double-stranded DNA, and red when combined with single stranded DNA (denaturated). Thawed spermatozoa were diluted with TNE buffer to 2 106 cells/mL. Samples were flash frozen in LN2 and stored at 80 °C until analysis. We calculated the DNA fragmentation Index (DFI) for each spermatozoon as the ratio of red fluorescence respect to total fluorescence (red + green). High values of DFI indicate chromatin abnormalities. The % DFI was calculated as the percentage of spermatozoa with DFI >25.
Semen preservation Semen preservation was performed in Experiment 2 using two different protocols. Thus, it was tested the effect of vitrification (direct immersion into liquid nitrogen) and it was compared to conventional cryopreservation protocol (cooling to 5 °C for 2 h and equilibration at this temperature for 2 h; total cooling time 4 h). Conventional cryopreservation was performed using the commercial extender BiladylÒ and two final sperm concentrations were reached: (i) a conventional of 200 106 spermatozoa/mL, and (ii) a concentration of 20 106 spermatozoa/mL, similar to that used in the vitrification procedure. This type of cryopreservation was carried out in two steps. Firstly, non-glycerolated fraction was added at 30 °C and the diluted semen (400 or 40 106 spermatozoa/mL)
was cooled to 5 °C for 2 h. Then, it was further diluted (v:v to a final concentration of 200 or 20 106 spermatozoa/mL) with the fraction containing glycerol (7%). The diluted sperm samples were then held at 5 °C for 2 h more before freezing (equilibration time) and after that the diluted semen was loaded into 0.25 mL plastic straws and frozen on over nitrogen vapors for 10 min, by placing the straws 4 cm above the surface of nitrogen liquid. The straws were subsequently plunged into liquid nitrogen and stored. For vitrification, different extenders were used: BiladylÒ at a final concentration of 200 or 20 106 spermatozoa/mL and combinations of sucrose and glycerol in a SOF-based media (final sperm concentration 20 106 spermatozoa/mL) in the following way: 0.03 M sucrose-3% glycerol, 0.03 M sucrose-7% glycerol, 0.05 M sucrose-3% glycerol and 0.05 M sucrose-7% glycerol. The SOF media used in this study was that described by Takahashi and First [40] without amino acids and supplemented with HEPES and 1% BSA. The diluted semen (100 lL) with different extenders was loaded into 0.25 mL straws and they were inserted into 0.5 mL straws which were heat-sealed and directly plunged into liquid nitrogen.
Statistical analyses Data preparation and statistical analyses were conducted using the R statistical packages [32]. Previous to data analysis, assumption of normality was tested and if not satisfied log or arcsin transformation was used. In Experiment 1, the effects of sucrose and glycerol concentrations at 3 times of incubation on sperm quality
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
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were examined using a linear regression model. The concentrations of sucrose and glycerol given the best quality on Experiment 1 were then used jointly in Experiment 2. Thus, a linear regression analysis was performed to examine the effects of extenders’ composition on sperm quality at thawing. Differences among extenders within each cryopreservation procedure were tested using a multiple comparisons analysis considering the Bonferroni adjustment. In all analyses, a threshold value of P < 0.05 was established to denote statistical significance.
Results Experiment 1: Effect of cytotoxicity of sucrose and glycerol at different concentrations on sperm quality after incubation period at 37 °C Samples extended in BiladylÒ showed higher sperm quality (P 6 0.05) than the other treatments evaluated including the control using only SOF (Fig. 2A and B; Tables 1 and 2 in Supplementary material). There was no effect of the dilution (P > 0.05), showing B50 and B5 similar values of sperm quality. Adding sucrose or glycerol had a significant negative effect (P 6 0.05) on sperm quality for all sperm parameters assessed, except for DFI which was unaffected. Moreover, higher sucrose or glycerol concentrations provided a decrease in sperm quality (P 6 0.05) (Fig. 2A and B; Tables 1 and 2 in Supplementary material). Throughout the incubation period (from 0 to 4 h), treatments except BiladylÒ controls showed a progressive decrease (P 6 0.05) of sperm quality, which was more noticeable for samples containing higher concentrations of sucrose or glycerol (Fig. 2A and B; Tables 1 and 2 in Supplementary material).
Experiment 2: Effect of vitrification on sperm quality after thawing The sperm quality was higher for samples cryopreserved in a way conventional in relation to vitrification procedure (Table 1; Fig. 1 in Supplementary material). There were no differences (P P 0.05) in the conventional cryopreservation between freezing at 20 or 200 106 spermatozoa/mL, although the sperm motility was slightly above for cryopreserved samples with the highest concentration. The samples vitrified showed a low sperm quality (P 6 0.05) when combinations of sucrose and glycerol were used, although the samples vitrified with BiladylÒ at 20 106 spermatozoa/mL showed acceptable values for the most of sperm parameters, except for motility. Thus, for this treatment the values of live spermatozoa with intact plasma membrane (YO-PRO-1-/PI-) and live spermatozoa with intact acrosome (PNA-/PI-) were higher (P 6 0.05) than for the rest of extenders (Table 1; Fig. 1 in Supplementary material).
Discussion Up to date, sperm cryopreservation in small ruminants has been performed by the conventional technique which involves three phases: cooling to 5 °C, equilibration at this temperature for a variable time and freezing on liquid nitrogen vapors. For this, extenders based on different concentrations of egg yolk and glycerol have been widely used. Conventional techniques have been well investigated and it has been shown that intra- or extra-cellular ice crystal formation and osmotic changes may occur during a slow freezing which led to extensive cell shrinkage and sperm damage [7,41]. As alternative, vitrification has been proposed as a
Fig. 2. Effect of sucrose and glycerol cytotoxicity on spermatozoa under incubation conditions. Data are means ± sem. B5: BiladylÒ at 20 106 spz/mL; B50: BiladylÒ at 200 106 spz/mL; 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), http://dx.doi.org/10.1016/j.cryobiol.2015.05.004
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P. Jiménez-Rabadán et al. / Cryobiology xxx (2015) xxx–xxx Table 1 Effect of extender and sperm concentration on seminal quality after thawing in vitrified and conventionally cryopreserved samples. Conventional cryopreservation
B50 B5 0.03 M-3% 0.03 M-7% 0.05 M-3% 0.05 M-7%
Vitrification
SM
YO-PRO1-/PI-
PNA-/PI-
DFI
SM
YO-PRO1-/PI-
PNA-/PI-
DFI
34.65 ± 9.53 16.60 ± 9.53
20.14 ± 4.65 30.66 ± 4.65
20.73 ± 4.00 31.34 ± 4.00
5.38 ± 1.07 5.75 ± 1.07
0.10 ± 0.05 0 ± 0.05 0 ± 0.73 0 ± 0.73 0 ± 0.73 0 ± 0.73
1.93 ± 2.09b 12.78 ± 2.09ª 1.29 ± 2.95b 0.91 ± 2.95b 0.77 ± 2.95b 0.24 ± 2.95b
1.76 ± 1.94b 10.09 ± 1.94ª 1.15 ± 2.74b 1.16 ± 2.74b 0.37 ± 2.74b 0.67 ± 2.74b
4.11 ± 0.80 6.08 ± 0.80 5.40 ± 1.13 4.71 ± 1.13 4.64 ± 1.13 4.00 ± 1.13
Data are means ± standard error. B50: BiladylÒ at final concentration of 200 106 spermatozoa/mL; B5: BiladylÒ at final concentration of 20 106 spermatozoa/mL; 0.03 M3%: 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).
procedure that avoids the formation of ice crystal which leads to some advantages in relation to conventional sperm cryopreservation. Moreover, vitrification can be performed using extenders without egg yolk or permeable cryoprotectants avoiding the negative effects of these substances [11,13] and using proteins and sugars as non-permeable extracellular cryoprotectants [19]. Focusing on animal sperm vitrification, few studies have achieved good results, so now efforts are being put in the design of new protocols which take into account new technologies and the use of different extenders as a way to increase the success of the methodology. And more specifically for sheep, there is little information available in relation to the use of alternative methods to preserve semen. For that reason, this study was firstly aimed to evaluate the toxicity of different cryoprotectants used for vitrification in other species (sucrose and/or glycerol) based on its concentration under incubation conditions. Subsequently, it was studied the effect of vitrification on the sperm quality at thawing when a combination of extenders at the concentrations which provided better results in the previous experiment were used. In the first experiment, there was no differences in sperm quality between controls B50 and B5, being the latter highly diluted. Contrary to our study, it has been showed that high dilutions of the sperm samples lead to damage of spermatozoa [22,24]. However, many assisted reproductive techniques, as sex-sorting, are performed using samples highly diluted without lack of sperm quality adding protector substances as egg yolk or seminal plasma [24]. In our study, the B5 control was used to mimic the conditions of vitrification, since the most of authors vitrify with low concentration and volume [1,3,26,37] and in this way to reject the hypothesis of a possible damage due to dilution. In relation to sucrose toxicity, our study showed how an increase of this sugar produced a reduction in sperm quality through the incubation period. Similar trend was observed when concentration of glycerol increased. Thus, samples with 0.03 and 0.05 M sucrose and 3% and 7% glycerol showed higher percentage of motile spermatozoa, viable spermatozoa and spermatozoa with intact acrosome. Contrary, higher concentrations of sucrose (0.15 M and 0.25 M) and glycerol (14% and 18%) resulted in lower sperm quality. In human, canine and rabbit, 0.25 M concentrations of sucrose had not a toxic effect on spermatozoa [14,36,37]. It has been described that sugars have a protector effect against osmotic changes decreasing the ice formation and stabilizing the solute concentration inside of sperm cell during osmotic stress as well as, have the property of stabilizate the cell membrane [19,20,29]. Moreover, a synergistic effect between sugars and permeable cryoprotectans has been reported in ram [28]. Therefore, the use of sugars in extenders could result in a decrease of concentration of permeable cryoprotectants used, thus reducing their negative effects on spermatozoa during the vitrification. Based on these observations, in a second experiment we examined in a vitrification procedure the effects of using extenders
based on combinations of sucrose and glycerol at those concentrations which provided the best results in the previous experiment. Results showed a decrease in the sperm quality after vitrification for extenders with combinations of 0.03 M and 0.05 M of sucrose with 3% and 7% of glycerol, respectively. Contrary to our results, the vitrification was carried out in human spermatozoa with good results [12]. Nawroth et al. [30], who vitrified human spermatozoa using extenders with and without cryoprotectans, observed the best results when samples were vitrified with free-cryoprotectans extenders. Similarly, the removal of permeable cryoprotectans from the vitrification media caused an improvement on the cryosurvival of fish and dog spermatozoa [26,37], although Agha-Rahimi et al. [1] did not found differences in the sperm quality of vitrified human spermatozoa using extenders with and without cryoprotectans. The success in the vitrification has been more limited for others species. Thus, in rabbit [36] and kangaroo [25] the sperm vitrification led to very few motile and/or viable cells post-thawing. The differences between studies could be due to the species. Shape and size of the sperm head and the flagellum are factors that define the cryosensivity of the cell [4,33,35]. A small size of head has been related with a higher cryoresistance in some species [4]. Thus, higher cryoresistance of vitrified spermatozoa in human could be due to the small size of the sperm head in relation with others species [8]. In the other hand, our results showed that vitrification offered best protection to sperm plasma and acrosomal membrane of spermatozoa in samples more diluted using egg yolk-glyerol based extender than in those samples vitrified with higher sperm concentration. However, the sperm motility was strongly affected in both conditions of concentration. It has been broadly assumed that the high dilution of the sperm samples lead to damage of spermatozoa [22], although Lehay et al. [21] showed that the high pre-freezing dilution at 20 106 spermatozoa/mL improved the viability, acrosome integrity and total motility after thawing in relation to samples more concentrated. The most authors have used low sperm volume and concentration to vitrify human spermatozoa with good results [3,26,37]. In addition, Maxwell and Jonhson [24] showed that egg yolk offers some protection to diluted spermatozoa. It is possible that the joint action of egg yolk and low sperm concentration used in the vitrification of ram spermatozoa would lead to higher protection of the spermatozoa. The lack of positive results in our study could be due not only to the technique of vitrification or extenders, but also other factors that were not considered, as the thawing rate. Recently, Mansilla et al. [23] found an influence of thawing temperature and time on sperm quality in vitrified samples, with better values for the highest temperature and shortest warming time. In our study the straws were thawed at 37 °C for 30 s being this combination very similar to that that showed the worst results in the previous study, 38 °C for 10 s [23].
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
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P. Jiménez-Rabadán et al. / Cryobiology xxx (2015) xxx–xxx
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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