Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants

Animal Reproduction Science 163 (2015) 75–81

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Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants Zhuangyuan Wu a , Xinbiao Zheng b , Yongming Luo b , Fei Huo b , Hong Dong b , Guoting Zhang b , Weihao Yu c , Fang Tian a , Liangjun He a , Jingbo Chen a,b,∗ a b c

College of Animal Science and Technology, Shihezi University, Shihezi 832000, Xinjiang, China Institute of Animal Science, Xinjiang Academy of Animal Science, Urumqi 830000, Xinjiang, China The Breeding Center of Yili Horse, Zhaosu 835600, Xingjiang, China

a r t i c l e

i n f o

Article history: Received 30 May 2015 Received in revised form 26 September 2015 Accepted 30 September 2015 Available online 3 October 2015 Keywords: Frozen semen Equine spermatozoa Horse Cryoprotective agents CPA

a b s t r a c t The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess whether this improved post-thaw spermatozoa quality. Gly, MF and DMF, were used to prepare seven combinations of freezing extender with different mixtures of cryoprotectant, and the 3.5% Gly, MF and DMF were used as a control group. The results show that post-thaw total motility, progressive motility, viability, and mitochondrial membrane potential for all concentrations of EG and DMSO were less than the 3.5% and 5% Gly and MF and 2.5% and 3.5% DMF (P < 0.05). Use of the 3.5% concentration resulted in the greater post-thaw total motility and progressive motility than the 2.5% and 5% concentrations for all CPAs. The results for the use of different combinations of cryoprotectant indicate there are differences in progressive motility and viability. The viability with the use of Gly(2/3) + MF(1/3) was 44.65% and was greater than the Gly(1/3) + MF(1/3) + DMF(1/3) (30.96%), MF(2/3) + DMF(1/3) (35.05%), Gly (32.21%) and MF(33.76%) (P < 0.05). The progressive motility with the use of the MF(2/3) + Gly(1/3) combination was 36.0% and was greater than in the DMF (25.0%) and MF(2/3) + DMF(1/3) (22.7%) (P < 0.05). These results suggest that using the appropriate cryoprotectant combination instead of a single cryoprotectant can improve horse spermatozoa cryopreservation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In modern horse breeding, frozen semen is popular because of its advantages compared to cooled-shipped

∗ Corresponding author at: College of Animal Science and Technology, Shihezi University, Shihezi 832000, Xinjiang, China. E-mail address: [email protected] (J. Chen). http://dx.doi.org/10.1016/j.anireprosci.2015.09.020 0378-4320/© 2015 Elsevier B.V. All rights reserved.

semen. The semen from a large part of the stallion population, however, cannot be used for semen freezing because of unsatisfactory post-thaw spermatozoa quality and fertility rates (Alvarenga et al., 2004; Vidament et al., 2002; Samper and Morris, 1998). Furthermore, large variations in semen quality after the freezing–thawing process and varying pregnancy rates after insemination occur between different stallions and ejaculates (Oehninger et al., 2000; Samper and Morris, 1998). Therefore, the

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continuing development of artificial insemination techniques with frozen-thawed spermatozoa in the equine industry requires the improved quality of frozen samples and the minimization of individual variability among stallions. In 1949, Polge et al. reported as a result of the “chance” discovery of the cryoprotective function of Gly during efforts to preserve avian spermatozoa in the frozen state. This report identified key elements that have an important role in the field of bio-preservation, including the need for a CPA (Polge et al., 1949). Gly has become widely used as a CPA for the frozen spermatozoa of various animals. In particular, the use of frozen bull spermatozoa has been a great success. However, the use of Gly has not been as successful for the cryopreservation of stallion spermatozoa, and a great variability of success is observed between individual stallions (Guay et al., 1981). Based on experimental studies of stallion spermatozoa, the most important factors causing cryoinjury are the toxicity caused by unequal distribution of CPAs, such as Gly, and the osmotic stress caused by dehydration of the extender and the cells during freezing and again at thawing (Morris et al., 2007; Morris, 2006). The cryoprotective capacity of any cryoprotectant agent varies widely across cell and tissue types (Karow, 1969); therefore, the efficacy of each cryoprotectant agent must be studied for each cell type. A large number of reports have been published regarding the freezing of ejaculated spermatozoa with several CPAs (Hoffmann et al., 2011; Nur et al., 2010; Medeiros et al., 2002; Morrier et al., 2002; Molinia et al., 1994). However, previous studies have provided inconsistent results. Neves et al. (1995) found that pregnancy rates were desirable with semen frozen using EG. Tracey (1999) evaluated five cryopotectants, Gly, EG, diethylene glycol, propylene glycol and DMSO at four molarities (0.5, 1.0, 1.5 and 2.0 mol/l) on multiple ejaculates from seven fertile breeding stallions. Overall, use of DMSO provided for the most desirable post-thaw motility and viability values and superior post thaw results for stallions that had semen for which use of Gly resulted in poor freezing capacity. Other studies used amides as cryoprotectants for stallions, with more significant improvements for semen from stallions with semen that could not be effectively frozen using Gly (Alvarenga et al., 2005). Some studies have revealed a significant improvement of the fertility of stallion semen frozen with DMF compared to Gly (Medeiros, 2003; Moffet et al., 2003). By contrast, Mantovani et al. (2002) report that use of EG reduced the percentage of motile and progressive motility for spermatozoa after thawing as compared with use of 3% Gly. Spermatozoa frozen using Gly as the cryoprotectant had greater percentages of motile and progressively motile spermatozoa compared to spermatozoa frozen using DMF (Moore et al., 2006). Use of MF and DMF protect stallion spermatozoa from cryodamage as effectively as Gly (Squires et al., 2004). Other experiments showed no differences in fertility rates for mares inseminated with semen that was frozen with extenders containing DMF or Gly. For example, daily inseminations of mares resulted in pregnancy rates of 46% and 50% for semen processed with freezing extenders containing 2% Gly or 2% DMF, respectively (Vidament et al., 2002).

The objective of the present study is to explore the effects of various CPAs and cryoprotectant combinations in the INRA96 extender on the plasma, acrosomal, mitochondrial membranes and motility of frozen-thawed stallion semen. 2. Materials and methods 2.1. Stallions and semen collection A total of six adult Yili stallions with proven fertility were housed at The Breeding Center for Yili Horses, Xingjiang China, and four horses were used for Experiment 1 and five for Experiment 2. Semen was collected from these stallions in September on a regular basis (two collections each week) using an artificial vagina. Six ejaculates per stallion were processed. After collection, raw semen was filtered to remove the gel portion. 2.2. Semen freezing and thawing Filtered semen of each ejaculate was diluted (1:1) in INRA96, consisting of a HGLL solution containing Hank’s salts, glucose and lactose, buffered with HEPES and supplemented with a purified fraction of native phosphocaseinate (Batellier et al., 1997). Extended samples were cooled at 22 ◦ C for 10 min and then centrifuged at 600 × g for 10 min. After centrifugation, the pellet was re-suspended in INRA96 supplemented with 2.5% clarified egg yolk to obtain 200 × 106 spermatozoa cells/ml. Then, 15 ml of resuspended semen was pooled from every stallion and the pooled sample was allocated to Experiments 1 or 2. In Experiment 1, re-suspended semen was aliquoted into 15 samples. Each sample was diluted with INRA96 containing 2.5% clarified egg yolk and 5%, 7%, or 10% of one of five different CPAs (Gly, EG, DMSO, MF and DMF). This resulted in final concentrations of 100 × 106 spermatozoa/ml and 2.55, 3.55 and 5% CPA. In Experiment 2, the semen was aliquoted into 10 batches and diluted with an equal amount of INRA96 containing 2.5% clarified egg yolk and two-fold the final concentration of desired additional cryoprotectant combinations (Table 1). The semen was loaded into 0.5 ml polyvinyl chloride straws (IMV-Technologies, L’Aigle, France), sealed with a filling and sealing machine (IMV-Technologies, L’Aigle, France), and then cooled to 4 ◦ C over 75 min. Freezing was performed with a programmable freezer (nitrogen freezer, automatic Mini-Digitcool, IMV-Technologies, L’Aigle, France) (−60 ◦ C/min until −140 ◦ C). The straws were stored in liquid nitrogen and then thawed in a water bath at 37 ◦ C for 30 s immediately before semen analyses. 2.3. Assessment of motility The motility parameters were evaluated using computer-assisted analyses (CEROSII, version 13, Hamilton Thorne, Beverly, MA, USA). For each analysis, 5 ␮l of diluted semen was mounted on a disposable Leja counting chamber (fixed height of 20 ␮m, Orange Medical, Brussels,

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Table 1 Composition of different freezing extenders. Groups Gly + MF (GM) MF + Gly (MG) Gly + DMF (GD) DMF + Gly (DG) MF + DMF (MD) DMF + MF (DM) Gly + MF + DMF(GMD) Gly MF DMF

Base extender

Gly (ml)

MF (ml)

2.3 4.7 2.3 4.7

4.7 2.3

INRA-96 + 5% clarified egg yolk(m/v) 93 ml

Belgium) and maintained at 37 ◦ C using a minitherm warmer stage for 2 min. The variables analyzed were total motility (average path velocity greater than 10 ␮m/s and VAP threshold for static cells are 10 ␮m/s), progressive motility (average path velocity greater than 30 ␮m/s and straightness of track greater than 80%). Analyses were performed for three observations/straw and three straws for each sample.

2.4. Assessment of plasma membrane integrity, acrosome integrity and mitochondrial membrane potential Spermatozoa membrane integrity was measured with the Live/Dead spermatozoa viability kit (Invitrogen, L7011). The thawed samples were diluted in PBS (1:1) and then centrifuged at 600 × g for 10 min. After centrifugation, the pellet was re-suspended in HEPES-buffered saline solution containing bovine serum albumin (10 mM HEPES, 150 mM NaCl, and 10% BSA, pH 7.4). A volume of 5 ␮l SYBR14 (0.02 mM) was added to a 1 ml sample of diluted semen, resulting in a final SYBR14 concentration of 100 nM. The vial was incubated in a darkened room for 10 min at 37 ◦ C and then 5 ␮l of propidium iodide (2.4 mM) was added and incubated an additional 8 min. After incubation, three slides were observed with a fluorescence microscope (OLYMPUS, ONIX992-IX71) with blue excitation (450–480 nm). At least 100 cells were counted per slide (300 cells/sample). The cells stained green were classified as viable, and red stained cells were classified as nonviable (Fig. 1). These results are presented as percentage of viable cells. Acrosome integrity was assessed using FITC-PNA (lectin agglutinin of Arachis Hypogaea, L-7381, Sigma Chemical, St. Louis, MO, USA). The thawed samples were diluted in PBS (1:1) and then centrifuged at 600 × g for 10 min. After centrifugation, the pellet was re-suspended in PBS. A volume of 20 ␮l FITC-PNA (1.125 mg/ml) was added to a 1 ml sample of diluted semen and incubated for 10 min in a water bath at 37 ◦ C. Three slides were observed with a fluorescence microscope (OLYMPUS, ONIX992IX71) with blue excitation (450–480 nm). Each microscopic field was evaluated first under fluorescence microscopy (40× magnification) to determine the number of defective acrosome spermatozoa and then under phase-contrast microscopy to determine the total number of spermatozoa per field (Fig. 1). At least 100 cells were counted per slide (300 cells/sample).

2.3 7

2.3 4.7 2.3

DMF (ml)

4.7 2.3 4.7 2.3 2.3

7 7

Mitochondrial membrane potential was assessed using JC-1 (Invitrogen, M34152). The thawed samples were diluted in PBS (1:1) and then centrifuged at 600 × g for 10 min. After centrifugation, the pellet was re-suspended in PBS. A volume of 10 ␮L JC-1 (200 ␮M) was added to a 1 ml sample of diluted semen and incubated for 20 min in a water bath at 37 ◦ C. Three slides were observed under a fluorescence microscope (OLYMPUS, ONIX992-IX71) with blue excitation (450–480 nm). Each microscopic field was evaluated first under fluorescence microscopy (40× magnification) to determine the number of high membrane potential spermatozoa and then under phase-contrast microscopy to determine the total number of spermatozoa per field (Fig. 1). At least 100 cells were counted per slide (300 cells/sample). 2.5. Statistical analysis Comparisons of the post-thaw total motility, progressive motility, viability, high membrane potential and defective acrosomes were conducted with ANOVA SPSS 13. Differences between means of parameters were subjected to an analysis of variance using the Tukey’s test, with P < 0.05 considered significant. 3. Results There were distinct differences in total and progressive motility using various CPAs (Table 2). Spermatozoa motility using the 3.5% Gly and all concentrations of MF and DMF was greater than with use of the 2.5% Gly and with use of all concentrations of DMSO and EG (P < 0.05). Progressive spermatozoa motility with use of the 2.5% and 3.5% MF was greater than with use of the 2.5% and 5% Gly, 5% DMF and with use of all concentrations of DMSO and EG (P < 0.05). Spermatozoa viability with use of the 3.5% MF and 5% Gly was greater than with use of the 2.5% Gly and with us of all concentrations of DMSO and EG (P < 0.05). Spermatozoa mitochondrial membrane potential with use of the 3.5% and 5% Gly and MF and 2.5% and 3.5% DMF was greater than with use of all concentrations of DMSO and EG (P < 0.05). There was no significant difference with the use of Gly, DMSO or EG for defective acrosomes (P > 0.05). The results (Table 3) of the combination cryoprotectant experiment indicate there were differences in progressive motility and viability (Table 2). Spermatozoa viability with use of GM was greater than with use of GMD, MD, and

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Fig. 1. Representative patterns of equine spermatozoa observed using STRA-14/PI, JC-1 and FITS-PNA staining (×40). Row 1: The spermatozoa stained green are viable and the red stained cells are nonviable (staining by STRA-14/PI). Row 2: A: JC-1 stained spermatozoa with high membrane potential; B: View of A under phase contrast microscopy. Row 3: A: FITC-PNA stained spermatozoa with defective acrosomes, B: View of A under phase contrast microscopy.

MF (P < 0.05). Spermatozoa progressive motility with use of MG was greater than with use of DMF and MD (P < 0.05). No significant differences were observed for spermatozoa post-thaw total motility, membrane potential and defective acrosomes (P > 0.05). 4. Discussion Penetrating CPAs are generally more effective than nonpenetrating CPAs because of the affect on several cell and solution properties. First, penetrating CPAs replace water inside the cell. This dehydrates the cell and the compounds inhibit the formation of intracellular ice. In addition, these CPAs increase the volume of the unfrozen channels

between the extracellular ice crystals, thereby increasing the space available for the cells. Furthermore, the CPAs decrease the salt concentration in the unfrozen solution (Meryman, 2007). However, because all the molecules permeate the cell plasma membrane more slowly than water, the CPAs induce transient somatically driven cell size changes that can damage a cell (Hammerstedt et al., 1990). In addition, the chemical toxicity of the cryoprotectant also causes spermatozoa damage (Gao et al., 1995). Therefore, the concentration and type of CPA influence the success of cryopreservation (Fernández-Santos et al., 2006; Watson and Holt, 2001; Salamon and Maxwell, 2000; Leibo, 1999). In the present study, use of 2.5%, 3.5% and 5% EG, DMSO, MF, DMF, and Gly were tested to determine the

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Table 2 Post-thaw total motility, progressive motility, viability, defective acrosome, and mitochondrial membrane potential (High m) of spermatozoa frozen (Mean ± SEM) in INRA96 supplemented with either 2.5%, 3.5% or 5% Gly, EG, DMSO, MF or DMF. CPAs GLY

EG

DMSO

MF

DMF

MOT (%) 2.5% 3.5% 5% 2.5% 3.5% 5% 2.5% 3.5% 5% 2.5% 3.5% 5% 2.5% 3.5% 5%

52.33 65.67 57.67 34.67 43.33 41.67 36.33 42.00 45.67 67.33 69.00 69.00 67.67 74.33 74.00

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

PMOT (%) c,d

2.08 2.52e,f 3.79d,e 5.03a 3.51a,b,c 7.09a,b 1.53a,b 1.00a,b 3.51b,c 2.31f 3.61f 4.00f 4.73f 3.21f 3.61f

23.00 30.00 24.33 15.00 19.00 18.67 15.33 17.67 18.33 34.67 34.00 30.67 29.67 30.67 23.00

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

High m (%)

Viability (%) b,c,d

1.00 3.61d,e 2.52c,d 2.65a 4.58a,b 2.31a,b 2.52a,b 1.53a,b 1.53a,b 2.52e 3.46e 3.79d,e 2.08d,e 0.58d,e 3.46b,c,d

27.26 32.26 35.87 7.12 6.39 5.50 7.75 5.28 5.70 30.48 35.27 30.11 32.63 29.32 30.24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.97 0.65b,c 2.37c 2.09a 0.72a 0.43a 1.79a 1.00a 0.63a 4.21b,c 1.84c 3.77b,c 2.40b,c 5.24b,c 3.62b,c

28.28 36.27 32.68 15.08 15.44 11.29 18.87 16.49 19.56 29.26 36.07 37.93 36.61 28.75 30.83

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Defect. Acro. (%)

b,c

5.59 2.84c 5.53c 1.79a 1.81a 1.27a 1.51a,b 3.41a 2.13a,b 0.16b,c 7.32c 6.99c 3.59c 1.44c 3.32b,c

33.26 43.00 41.77 39.25 34.78 45.08 51.63 47.85 43.73 45.56 39.36 38.67 34.85 41.82 45.50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.26 7.99 5.14 5.38 2.65 7.58 7.00 5.73 5.91 6.14 8.08 8.34 4.70 6.21 15.61

Different superscripts differa b c d e f ; Comparison between groups (P < 0.05). MOT, total motility; PMOT, progressive motility; Defect. Acro., defective acrosome; High m, high mitochondrial membrane potential.

most appropriate type and concentration for frozen stallion spermatozoa. Results of the present study indicate that stallion spermatozoa frozen in EG and DMSO are less viable than cells frozen using the other three CPAs. This result is consistent with the findings of Moore et al. (2006) and Mantovani et al. (2002), but is different than the findings of Hoffmann et al. (2011) and Tracey (1999). Results of the latter experiment indicate using DMSO as a CPA results in enhanced post-thaw spermatozoa characteristics compared to use of Gly or EG. The results of Hoffmann et al. (2011) indicate there are no significant differences with use of 5% and 3% EG, Gly, MF and DMF for spermatozoa progressive motility and viability after thawing. This may because of the extender, freezing procedure and stallion. In the present study, INRA96 + 2.5% clarified egg yolk was used as a base extender, whereas Tracey (1999) and Hoffmann et al. (2011) selected lactose-yolk extender and INRA82 containing 2% egg yolk as a base extender, respectively. INRA96 is a chemically defined extender developed for fresh semen storage at 4 or 15 ◦ C (Batellier et al., 2001) and is also an excellent base extender for frozen horse spermatozoa. Scherzer et al. (2009) compared EZ Mixin and INRA96 + 2%

clarified egg yolk as a base extender for frozen stallion spermatozoa and results showed that the freezing effect with use of INRA96 was superior to use of EZ Mixin. Plodie et al. (2008) reported that pregnancy rate and spermatozoa membrane integrity were enhanced with semen preserved in INRA96 than INRA82. Although these previous studies indicate that use of INRA96 as a base extender in combination with Gly yields a beneficial cryoprotective effect, the results of the current study show that it is not suitable in combination with EG and DMSO. No significant differences in post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity of spermatozoa were found in the present study with use of the 3.5% and 5% Gly and all concentrations of MF and DMF. There was a slightly greater post-thaw and progressive motility for spermatozoa stored using MF or DMF in the present study. These data are consistent with previous reports (Hoffmann et al., 2011; Squires et al., 2004; Medeiros et al., 2002). This finding can be attributed to MF and DMF having greater permeability coefficients for spermatozoa than Gly resulting in less osmotic stress injury during freezing and thawing. A 3.5% concentration achieved a greater post-thawing total and

Table 3 Post-thawing total motility, progressive motility, viability, defective acrosomes, and mitochondrial membrane potential of spermatozoa frozen (Mean ± SEM) in INRA96 supplemented with 2.5% egg yolk and different cryoprotectant combinations. CPAs

MOT (%)

GM MG GD DG MD DM GMD Gly MF DMF

± ± ± ± ± ± ± ± ± ±

70.00 67.67 64.33 67.67 64.33 66.67 60.00 62.33 69.33 63.67

PMOT (%) 4.00 2.89 4.04 4.51 2.89 4.16 5.29 4.93 5.33 2.52

34.00 36.00 32.00 28.67 22.67 31.00 30.00 27.00 35.00 25.00

± ± ± ± ± ± ± ± ± ±

High m (%)

Viability (%) b,c

2.00 4.00c 1.73a,b,c 2.89a,b,c 3.21a 2.65a,b,c 4.58a,b,c 3.00a,b,c 4.36b,c 5.29a,b

44.65 35.82 37.35 36.15 26.73 35.05 30.96 32.21 33.76 36.76

± ± ± ± ± ± ± ± ± ±

c

4.36 4.98a,b,c 1.38b,c 2.60a,b,c 2.85a 5.01a,b,c 1.75a,b 3.31a,b 4.20a,b 3.38a,b,c

37.87 36.38 30.89 27.01 38.46 36.61 37.80 30.41 41.64 43.62

± ± ± ± ± ± ± ± ± ±

0.88 7.17 2.11 8.03 0.36 2.34 11.24 7.77 3.60 4.71

Defect. Acro. (%) 39.82 36.38 47.20 51.68 37.72 44.97 38.88 40.68 47.15 34.94

± ± ± ± ± ± ± ± ± ±

5.87 7.17 5.03 5.41 10.28 3.50 5.98 3.00 10.21 3.32

Different superscripts differa b c d e f ; Comparison between groups (P < 0.05). MOT, total motility; PMOT, progressive motility; Defect. Acro., defective acrosome; High m: high mitochondrial membrane potential. GM: Gly(2.3%) + MF(4.7%); MG: MF(2.3%) + Gly(4.7%); GD: Gly(2.3%) + DMF(4.7%); DG: DMF(2.3%) + Gly(4.7%); MD: MF(2.3%) + DMF(4.7%); DM: DMF(2.3%) + MF(4.7%); GMD: Gly(2.3%) + MF(2.3%) + DMF(2.3%); Gly: glycerol(7%); MF: methyl formamide (7%); DMF: dimethylformamide (7%).

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progressive motility of spermatozoa than with use of the 2.5% and 5% concentrations for all CPAs. The cryoprotective capacity of CPA is dependent upon the permeability coefficients, the number of lone-pair electrons in the compound, the solubility in water (Nash, 1996) and the impact of CPA on cell membrane structure. EG and amides of low molecular weight with high permeability coefficients (Alvarenga et al., 2005; Medeiros et al., 2002) cross the cellular membrane more rapidly, limiting the exposure to osmotic stress. Gly stabilizes membranes (Almid et al., 1988; Alvarez and Storey, 1993) which may explain the greater survival with cryopreservation using Gly. Use of DMSO resulted in greater hydraulic conductivity for stallion spermatozoa compared to use of the other agents (Tracey, 1999). When the different chemical natures of CPAs are considered, a certain type of CPA may be more desirable than others for a particular characteristic. It is, therefore, proposed that freezing stallion spermatozoa with a combination of CPAs can take advantage of the varying desirable qualities of different CPAs and ultimately result in an improved cryoprotective effect. To verify this hypothesis, Gly, MF and DMF were selected for the present study and desirable results for spermatozoa viability were achieved in Experiment 1 using seven types of freezing extenders containing different combinations of CPAs (Table 1). The experiment demonstrates that the cryoprotectant combination of Gly and MF yields a beneficial cryoprotective effect compared to the control groups. These results suggest that using the appropriate cryoprotectant combination instead of a single cryoprotectant improves stallion spermatozoa cryopreservation. The use of combinations of CPAs can be extended to other animals, particularly for animals in which frozen semen technology is not yet successful. Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This research was supported by the National Natural Science Foundation of China (31360550) and the National Science and Technology Support Program of China (2012BAD44B02). References Almid, T., Johnson, L.A., Clarke, R.N., Pursel, V.G., 1988. Cryopreservation of boar semen: studies to determine optimum glycerol levels and the relationship of in vitro valuation to in vivo fertility. In: 11th International Congress on Animal Reproduction and A. I, p. 220. Alvarenga, M.A., Leao, K.M., Papa, F.O., Landim-Alvarenga, F.e., Medeiros, A.S.L., Gomes, G.M., 2004. The use of alternative cryoprotectors for freezing stallion semen. In: Proceedings of a Workshop on transporting Gametes and Embryos, 2nd–5th October, 2003, Brewster, MA, R & W Publications.

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