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Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality
REPBIO 182 1–6 reproductive biology xxx (2016) xxx–xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio 1 2 3
Original Research Article
Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality
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Chung-Hsun Yang a, Ting-Wen Wu a, Feng-Pang Cheng b, Jiann-Hsiung Wang a, Jui-Te Wu a,c,* a Department of Veterinary Medicine, College of Agriculture, National Chiayi University, 580 Xinmin Road, Chiayi City, Taiwan b Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan c Animal Teaching Hospital, College of Agriculture, National Chiayi University, 580 Xinmin Road, Chiayi City, Taiwan
article info
abstract
Article history:
The current study aimed to investigate the effects of different concentrations of glycerol (0%,
Received 16 June 2015
1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm
Received in revised form
quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen
21 December 2015
was collected from Duroc boars and was allocated to 32 treatment groups for cryopreser-
Accepted 30 December 2015
vation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI)
Available online xxx
was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in
Keywords:
semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b)
Glycerol
dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in
Dimethylacetamide
the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference
Semen
observed among these three treatments. The farrowing rates after AI with post-thawed
Cryopreservation
semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the
Boar
results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa. # 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
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* Corresponding author at: Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi City, Taiwan. Tel.: +886 5 2732953; fax: +886 5 2732917. E-mail address: [email protected] (J.-T. Wu). http://dx.doi.org/10.1016/j.repbio.2015.12.008 1642-431X/# 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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The advantages of using cryopreserved boar semen include longterm preservation of favorable genetic resources, long-distance transportation of valuable genetic material and guaranty to prevent the spread of pathogens [1]. Boar spermatozoa are highly sensitive to the freeze–thaw process, which causes considerable cell damage and leads to substantial reductions in farrowing rates and litter size. These factors are responsible for the limited use of frozen boar semen at the commercial level [1,2]. Routinely, semen is diluted in an extender containing cryoprotectants in order to minimize intracellular crystallization. Glycerol is the most commonly used penetrating cryoprotectant for cryopreservation of mammalian semen and effectively preserves post-thaw sperm function (motility, viability and acrosome integrity) [3]. Despite its benefits, some studies have reported low fertility rates when boar spermatozoa were frozen with extenders containing a high concentration of glycerol (4–8%) [4–8]. Recently, several amides, such as dimethylacetamide (DMA) and dimethylformamide (DMF) have been used as an effective substitute for glycerol in the freezing protocols for boar, stallion, rooster and rabbit semen [7,9–11]. Since most amides are hydrophilic by nature and have a lower molecular weight compared to glycerol, they may induce a limited osmotic damage to spermatozoa [12]. In addition, diverse chemical structures and hydrophilic nature of cryoprotectants might trigger diverse reactions with the sperm cells that could affect post-thaw semen quality [13–16]. The freezing and thawing rates also influence sperm cryosurvival [17,18]. An optimal cooling rate must therefore be determined to minimize cryoinjuries, which include the disruption of the sperm plasma membrane and DNA structure, induced by extensive intracellular ice formation as well as changes in the intracellular pH and ionic composition [17,19,20]. Our previous study demonstrated that favorable straw freezing methods for glycerol-based extenders were different from those with DMA, and the interaction that occurred between the cooling velocity and cryoprotectants affected post-thaw boar semen quality [8]. Furthermore, the experimental design of the current study was principally derived from our previous research [8], which showed that the addition of Equex STM paste to extenders containing 5% DMA or 5% glycerol could improve the cryosurvival of boar spermatozoa, and highlighted the beneficial effects of the straw freezing method with different cryoprotectants. However, there were low fertility rates in preliminary artificial insemination (AI) trials using extenders containing 5% DMA or 5% glycerol. Therefore, the aim of the current study was to evaluate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and DMA (0%, 1%, 3%, and 5%) as well as the straw freezing method (liquid nitrogen or dry ice) on post-thaw boar semen quality.
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Introduction
2.
Materials and methods
2.1.
Semen collections
The gel-free fraction of boar ejaculate (approximately 200– 350 mL) was collected into a pre-warmed plastic container
(38 8C) covered with gauze, using the gloved-hand technique. The ejaculates were collected from two or three Duroc boars, giving a total of seven ejaculates. Sperm motility was evaluated subjectively with a phase contrast optical microscope (Olympus BX50, Tokyo, Japan) at 100 magnification. Only ejaculates with ≥70% motility were processed. Sperm concentration was calculated, using a haemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) [21].
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2.2.
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Semen cryopreservation
Immediately after collection, the ejaculates were diluted (1:1; v/v) in Beltsville Thawing Solution extender (BTS; Minitüb, Abfüll-und Labortechnik GmbH & Co. KG, Tiefenbach, Germany) [22] and transferred to the laboratory in a container (16 8C) within 60 min. On arrival at the laboratory, each extended semen sample was immediately centrifuged at 800 T g for 10 min at 16 8C. The supernatant was discarded and the sperm pellets were re-suspended (1:1; v/v) in a lactoseegg yolk extender (11% lactose solution (w/v) and 20% egg yolk, 0.5% Equex STM paste). Subsequently, each semen sample was divided in 32 equal parts for the treatment, stored in a container for 60 min at 16 8C and then further cooled for 120 min at 5 8C. Following cooling at 5 8C, the freezing extender containing glycerol (Sigma, St. Louis, MO, USA) and/or N,Ndimethylacetamide (DMA, Sigma) was added to the samples. The freezing extenders consisted of different concentrations of glycerol (0, 1, 2, 3 or 5%) and/or DMA (0, 1, 3 or 5%) yielding 16 samples to be frozen in dry ice (D-treatments) and 16 samples to be frozen in liquid nitrogen (N-treatments). The specific concentrations of glycerol and DMA in each D- or N-treatment are depicted in first three columns of Tables 2 and 3. Semen sample was diluted (2:1; v/v) in the freezing extender and packaged in 0.5 mL straws (400–450 106 spermatozoa/mL). The first set of cooled 16 semen samples (D samples) was frozen by placing straws horizontally at the surface of dry ice for 10 min, whereas the second set of 16 samples (N samples) were frozen by placing the straws horizontally at 3 cm above the surface of liquid nitrogen for 20 min. At the end, all frozen straws were stored in liquid nitrogen until required. For sperm assessments, the frozen straws were thawed for 20 s at 37 8C, and the semen samples were re-suspended (1:20, v/v) in a prewarmed (37 8C) BTS extender.
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2.3.
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Semen evaluation
Ten microliters of diluted semen were placed on a microscopic slide, covered with a cover glass (18 mm 18 mm) and at least 10 microscopic fields were examined at 200 magnification under a phase contrast optical microscope (Olympus BX50) equipped with a pre-warmed (38 8C) stage. Sperm viability and morphology were studied using nigrosin and eosin [23]. Morphological abnormalities of spermatozoa were classified as major or minor [24]. Only major abnormalities were recorded when both major (e.g., teratoid head, kinked midpiece, double tails) and minor abnormalities (e.g., detached head, simple bent or coiled tail, terminally coiled tail) simultaneously occurred in a spermatozoon. Acrosome integrity of fixed spermatozoa was assessed using a fluorescein iosthiocyanate labeled peanut agglutinin
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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(FITC-PNA; E.-Y. Lab. Inc., San Mateo, CA, USA) [25] with some modifications [26]. Briefly, frozen-thawed spermatozoa were washed by centrifugation (600 g, 5 min) with phosphatebuffered saline (PBS) to remove non-sperm components and fixed in 0.5% glutaraldehyde and 2% paraformaldehyde (Sigma). After washing, the fixed spermatozoa were stained with 20 mg/mL FITC-PNA for 30 min at 37 8C. The washing procedure was repeated to remove the remaining FITC-PNA and subsequently counter-stained with propidium iodide (FertiLight® kit, Molecular Probes Inc. Eugene, OR, USA) for 10 min at 37 8C. Spermatozoa exhibited two major fluorescent patterns when observed under epifluorescence microscope (Nikon OPTIPHOT-2, Tokyo, Japan): (1) spermatozoa with intact acrosome lacked green fluorescence over the apical portion of the head region and equatorial segment, and (2) spermatozoa with damaged acrosome exhibited a distinct green fluorescence over the apical portion of the head region or equatorial segment. The acrosome integrity was assessed in at least 200 spermatozoa per sample. Sperm longevity was determined by calculating the proportions of motile and viable spermatozoa following a 6-h post-thaw incubation period at 37 8C.
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2.4.
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Fourteen multiparous sows were used for the AI experiment. Each sow was checked for heat twice a day. All sows were artificially inseminated at 24, 36 and 48 h after standing heat, and semen was delivered into cervix with a catheter [21]. The insemination was carried out by the same operator. Three and four sows were separately inseminated with post-thaw semen of 9D-treatment and 10N-treatment, respectively, whereas seven sows were inseminated with fresh semen diluted in BTS. The farrowing rate (%) and total number of piglets were recorded.
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Post-thaw sperm quality was analyzed in duplicate. The statistical analysis was performed using SPSS (SPSS, V12.0, SPSS Inc., Chicago, IL, USA). Before the analysis, normal distribution of data was confirmed. The percentage data were analyzed by generalized linear model (GLM) and incorporated an ANOVA factorial design to study the main effects of cryoprotectant (at various concentrations of glycerol and DMA), freezing method (dry ice and liquid nitrogen) and incubation time (0 and 6 h; only sperm motility and viability) and their interactions on the
Artificial insemination (AI)
Statistical analysis
analyzed post-thaw sperm parameters. If ANOVA showed significant differences among treatment means, a planned multiple comparison of means was examined by post hoc Tukey's test. Values are presented as the mean SEM, and differences were considered significant at p < 0.05.
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3.
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Results
The mean values of sperm motility, viability and acrosome integrity for fresh semen extended in BTS were 86%, 88% and 91%, respectively. The ANOVA results of the three main effects (cryoprotectant, freezing method and incubation time) for post-thaw semen quality are shown in Table 1. Cryoprotectant significantly affected post-thaw semen quality except for normal sperm morphology. Freezing method significantly influenced post-thaw sperm acrosome integrity and normal morphology, whereas incubation time had a significant effect on motility and viability of frozen-thawed spermatozoa. Among the sixteen D-treatments, the best scores for postthaw semen quality were observed in the 16D group (5% glycerol alone), with sperm motility, viability and acrosome integrity determined as 48 3%, 53 3%, and 52 1%, respectively (Table 2). There were no significant differences (p > 0.05) in post-thaw semen quality between the 16D group and the 9D (3% glycerol alone) group regardless of the incubation time. In addition to 16D and 9D groups, the 5D-, 10D-, 11D- and 15D-treatments also had beneficial effects on post-thaw semen quality in terms of sperm motility and viability (Table 2). Among the sixteen N-treatments, the best scores for postthaw semen quality were observed in the 10N group (3% glycerol, 1% DMA), with sperm motility, viability and acrosome integrity determined as 44 3%, 48 3%, and 45 1%, respectively (Table 3). There were no significant differences (p > 0.05) observed between the 10N group and 9N (3% glycerol alone) group regardless of the incubation time. Post-thaw sperm morphology did not differ (p > 0.05) among the examined groups either within the D-treatment (Table 2) or the N-treatment (Table 3). Preliminary AI trials showed that the farrowing rate and litter size obtained with using fresh semen were 86% and 10.2, respectively. The farrowing rates and litter sizes obtained in the 9D group and 10N group were 33% and 6, and 50% and 6.5, respectively.
Table 1 – ANOVA sources of variations in post-thaw boar semen quality. Sperm parameters Sources Cryoprotectant (C) Freezing method (F) Incubation time (I) CF CI FI
Motility
Viability
Acrosome integrity
Normal morphology
<0.001 >0.177 <0.001 <0.001 <0.001 >0.514
<0.001 >0.150 <0.001 <0.001 <0.001 >0.490
<0.001 <0.003 – <0.001 – –
>0.147 <0.012 – >0.137 – –
Significant at p < 0.05.
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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Table 2 – Effects of various concentrations of glycerol and/or DMA on post-thaw boar semen quality (mean W SEM) following freezing in dry ice. Treatment
Cryoprotectant Gly %
1D 2D 3D 4D 5D 6D 7D 8D 9D 10D 11D 12D 13D 14D 15D 16D
1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 5
Sperm parameters (%)
DMA %
0 1 3 5 0 1 3 5 0 1 3 5 1 3 5 0
0h
6h
Motility
Viability
Acrosome integrity
Normal morphology
Motility
Viability
26 2 cd 28 2 cd 33 3bcd 22 2 d 38 3 abc 34 3bcd 33 2bcd 24 2 d 47 4 a 42 2ab 38 1abc 32 2bcd 23 2 d 23 3 d 40 2ab 48 3a
28 3 cd 30 2 cd 35 3 bcd 23 2 d 41 4 abc 36 3 bcd 35 2 bcd 26 2 d 50 4 a 45 2 ab 41 1 abc 34 3 bcd 24 2 d 25 3 d 43 2 ab 53 3 a
29 1 fg 30 1 f 36 1 e 26 1 h 41 1 cd 38 1 de 37 1 e 28 1 fgh 50 1 a 46 1 b 40 1 cd 35 1 e 26 1 gh 27 1 gh 42 1 c 52 1 a
89 1 88 2 88 1 90 1 86 2 88 3 87 2 89 2 87 1 89 1 88 2 87 3 89 2 88 2 89 1 88 1
8 1 cd 11 1 bcd 9 1 cd 7 1 cd 12 1 abcd 13 3 abcd 9 1 cd 8 1 cd 18 3 ab 15 1 abc 10 1 cd 6 1d 6 1d 9 1 cd 11 3 abcd 19 2 a
9 1 cd 11 1 bcd 9 1 cd 8 1 cd 13 1 abcd 13 4 abcd 9 2 cd 9 1 cd 20 3 ab 16 1 abc 10 1 cd 7 1d 7 1d 10 1 cd 12 4 bcd 21 2 a
D – dry ice; Gly – glycerol; DMA – dimethylacetamide. Within a column, values with different letters differ significantly ( p < 0.05). n = 7 ejaculates.
Table 3 – Effects of various concentrations of glycerol and/or DMA on post-thaw boar semen quality (mean W SEM) following freezing in liquid nitrogen. Treatment
Cryoprotectant Gly %
1N 2N 3N 4N 5N 6N 7N 8N 9N 10N 11N 12N 13N 14N 15N 16N
1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 5
Sperm parameters (%)
DMA %
0 1 3 5 0 1 3 5 0 1 3 5 1 3 5 0
0h
6h
Motility
Viability
Acrosome integrity
Normal morphology
Motility
Viability
33 2abcde 22 1 e 28 2 cde 30 2 cde 35 2 abcd 34 3 abcde 28 3 cde 24 2 de 43 2 ab 44 3 a 33 1 abcde 31 3 bcde 32 3 abcde 28 2 cde 34 3 abcde 37 4 abc
35 2 abcde 23 1 e 30 2 cde 32 3 cde 38 2 abcd 37 3 abcde 29 3 cde 25 2 de 45 2 ab 48 3 a 35 2 abcde 34 3 bcde 34 3 abcde 29 3 cde 36 4 abcde 40 4 abc
38 1 bcd 28 1 h 33 1 fg 36 1 cde 40 1 b 38 1 bc 33 1 efg 28 1 h 44 1 a 45 1 a 32 1 fg 35 1 cdef 35 1 def 31 1 g 35 1 def 40 1 b
86 2 88 2 86 2 87 3 85 1 87 3 88 2 88 2 89 1 87 1 85 2 88 1 88 3 89 2 87 2 86 2
7 1 de 7 1 de 8 1 cde 6 1e 13 2 abcd 11 1 bcde 7 1 de 6 1 de 16 2 ab 19 1 a 9 1 cde 6 1 de 10 2 bcde 9 1 cde 14 2 abc 17 3 ab
8 1 de 7 1 de 8 1 cde 6 1e 14 2 abcd 12 1 bcde 8 1 de 7 1 de 18 3 ab 20 1 a 10 1 cde 7 1 ef 11 2 de 9 1 bcde 15 2 cde 18 3 abc
N – liquid nitrogen; Gly – glycerol; DMA – dimethylacetamide. Within a column, values with different letters differ significantly (p < 0.05). n = 7 ejaculates.
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The effects of two cryoprotectants with two straw freezing methods on post-thaw boar semen quality were examined in the current study. The results clearly demonstrated that the semen sample with extender containing 5% glycerol and
Discussion
frozen in dry ice (16D-treatment) yielded the highest mean values of post-thaw sperm motility, viability, and acrosome integrity compared with other treatments. Some reports have demonstrated low fertility rates when boar spermatozoa were frozen in extenders containing a high concentration of glycerol (4–8%) [4–6]. Corcuera et al. [6] reported that spermatozoa frozen in high concentrations of glycerol might compromise
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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the sperm acrosome integrity and increase chromatin condensation and stability. These increases, in turn, may induce early embryonic death or lower embryonic development [27]. Although we did not observed the detrimental effect of high glycerol concentrations on post-thaw sperm acrosome integrity in the current or previous studies [8]. However, low farrowing rates (0–8.3%) were evident in our preliminary AI trials with post-thawed semen frozen in extender containing 5% DMA or 5% glycerol (unpublished data). Interestingly, in the present study, the post-thaw semen qualities of extenders with 3% glycerol alone were similar to those with 5% glycerol alone (9D-treatment vs. 16D-treatment or 9N-treatment vs. 16N-treatment). However, this phenomenon was not observed when semen was preserved in extenders with DMA alone (14D-treatment vs. 15D-treatment or 14N-treatment vs. 15N-treatment). In the present study, the experimental procedure involved a modified protocol from that used in a previous study [8]. Unpublished data and previous research indicated that the cytotoxicity of high glycerol concentrations (4–8%) on fertility could be decreased by modifying the cryopreservation process (e.g., altering the freezing method and lowering cryoprotectant concentrations), and those modifications could further improve post-thaw sperm quality, such as fertilizing potential [4–8]. In the present study, we evaluated the fertilizing ability of frozen-thawed spermatozoa using a glycerol-based extender (9D-treatment) and extender containing both glycerol and DMA (10Ntreatment). Only 7 sows were used in AIs owing to financial limitations; however, the 9N-treatment and other treatments that had beneficial effects on sperm cryosurvival will be considered for further AI trials. In the present study, we also evaluated whether the combinations of different concentrations of two penetrating cryoprotectants (glycerol and DMA) could achieve similar or improve the cryosurvival of boar spermatozoa [4–6]. Other than the combinations of 1% glycerol and 1 or 3% DMA for samples frozen in dry ice (2D- or 3D-treatment), or 3% glycerol and 1% DMA for samples frozen in liquid nitrogen (10Ntreatment), DMA had no beneficial effects on sperm cryosurvival when added to the glycerol-based extender. This possibly indicates that the combination of glycerol and DMA did not significantly affect the cryosurvival of boar spermatozoa. Similar results were observed by Malo et al. [28], who demonstrated that DMF was less effective than glycerol in the cryopreservation of boar semen, when used alone or in combination with glycerol. Kim et al. [29] showed that DMA and dimethylsulfoxide (DMSO) were less effective than glycerol in improving post-thaw semen quality. Furthermore, Moustacas et al. [30] demonstrated that DMF had no beneficial effects on post-thaw quality of ram semen when used alone or in combination with glycerol. It should be emphasized that the penetrating cryoprotectants have unique structures and characteristics that could influence the mechanism of interaction with cellular structures, ultimately affecting sperm cryosurvival [15]. The results of the present study indicate that DMA might interfere with the interaction of glycerol and the sperm structure, intracellular ice formation, and osmolarity, which could induce increased cytotoxicity in sperm function. The present study compared the effects of two simple freezing methods on post-thaw semen quality. The results
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indicated that the straw-freezing method using dry ice was more favorable for the extender containing 5% glycerol or 5% DMA (15D- vs. 15N-treatment or 16D- vs. 16N-treatment). However, Wu et al. [8] demonstrated that the straw-freezing method using liquid nitrogen was more favorable for extenders containing 5% DMA. The major difference between the results was the higher sperm concentration used in the present study (800–900 106 sperm/mL) than in the previous study (150 106 sperm/mL). Although glycerol and DMA, as penetrating cryoprotectants, allow samples to be frozen immediately without incubation [6,8], high sperm concentrations may hinder the interaction of glycerol or DMA with the sperm structures. This could affect the rate of osmoticallydriven water flow, which may affect membrane permeability, intracellular pH, and ionic composition [17,19,20]. Therefore, prolonged incubation and slower freezing rates might have contributed to the beneficial effects in the present study and other studies [31]. Corcuera et al. [6] observed improved motility and acrosome integrity when boar spermatozoa were incubated in 2% glycerol-based extender for 30 min at 5 8C before cryopreservation. Since the freezing rate differed among treatments due to different spermatozoa and cryoprotectant concentrations or exposure time, a controlled-rate freezer was used to establish the optimal freezing rate, as discussed by Thurston et al. [32]. In conclusion, the results of the present study indicated that a combination of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa. However, the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the most effectual post-thaw quality parameters of boar semen.
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Conflict of interest
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All authors declare that they have no conflict of interests.
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[20] Hammadeh ME, Askari AS, Gerrg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl 1999;22:155–62. [21] Brillard JP, McDaniel GR. The reliability and efficiency of various methods for estimating spermatozoa concentration. Poult Sci 1985;64:155–8. [22] Garcia JC, Dominguez JC, Pena FJ, Alegre B, Gonzalez R, Castro MJ, et al. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160–5. [23] Blom E. A one-minute live-dead sperm stain by means of eosin–nigrosin. Fertil Steril 1950;1:176–7. [24] Oettle EE, Soley JT. Sperm abnormalities in the dog: a light and electron microscopic study. Vet Med Rev 1988;59:28–70. [25] Cheng FP, Fazeli AR, Voorhout WF, Marks A, Bevers MM, Colenbrander B. Use of PNA (peanut agglutinin) to assess the acrosomal status and the zona pellucida induced acrosome reaction in stallion spermatozoa. J Androl 1996; 17:674–82. [26] Farlin ME, Jasko DJ, Graham JK, Squires EL. Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa. Mol Reprod Dev 1992;32:23–7. [27] Lwoff L, Bézard J, Paquignon M. Comparison of conservation technologies of boar spermatozoa. Effect on the mechanisms of fertilization. J Rech Porcine Fr 1987;19: 79–86. [28] Malo C, Gil L, Cano R, Martínez F, García A, Jerez RA. Dimethylformamide is not better than glycerol for cryopreservation of boar semen. J Androl 2012;44:605–10. [29] Kim S, Lee YJ, Ji DB, Kim YJ. Evaluation of different cryoprotectants (CPAs) in boar semen cryopreservation. J Vet Med Sci 2011;73:961–3. [30] Moustacas VS, Cruz BC, Varago FC, Miranda DA, Lage PG, Henry M. Extenders containing dimethylformamide associated or not with glycerol are ineffective for ovine sperm cryopreservation. Reprod Domest Anim 2011;46:924–5. [31] Iaffaldano N, Iorio MD, Rosato MP. The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: dimethylacetamide versus dimethylsulfoxide. Theriogenology 2012;78:1381–9. [32] Thurston LM, Holt WV, Watson PF. Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison. Theriogenology 2003;60:101–13.
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Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio 1 2 3
Original Research Article
Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality
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Q1
Chung-Hsun Yang a, Ting-Wen Wu a, Feng-Pang Cheng b, Jiann-Hsiung Wang a, Jui-Te Wu a,c,* a Department of Veterinary Medicine, College of Agriculture, National Chiayi University, 580 Xinmin Road, Chiayi City, Taiwan b Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan c Animal Teaching Hospital, College of Agriculture, National Chiayi University, 580 Xinmin Road, Chiayi City, Taiwan
article info
abstract
Article history:
The current study aimed to investigate the effects of different concentrations of glycerol (0%,
Received 16 June 2015
1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm
Received in revised form
quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen
21 December 2015
was collected from Duroc boars and was allocated to 32 treatment groups for cryopreser-
Accepted 30 December 2015
vation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI)
Available online xxx
was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in
Keywords:
semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b)
Glycerol
dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in
Dimethylacetamide
the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference
Semen
observed among these three treatments. The farrowing rates after AI with post-thawed
Cryopreservation
semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the
Boar
results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa. # 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
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* Corresponding author at: Department of Veterinary Medicine, College of Agriculture, National Chiayi University, Chiayi City, Taiwan. Tel.: +886 5 2732953; fax: +886 5 2732917. E-mail address: [email protected] (J.-T. Wu). http://dx.doi.org/10.1016/j.repbio.2015.12.008 1642-431X/# 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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The advantages of using cryopreserved boar semen include longterm preservation of favorable genetic resources, long-distance transportation of valuable genetic material and guaranty to prevent the spread of pathogens [1]. Boar spermatozoa are highly sensitive to the freeze–thaw process, which causes considerable cell damage and leads to substantial reductions in farrowing rates and litter size. These factors are responsible for the limited use of frozen boar semen at the commercial level [1,2]. Routinely, semen is diluted in an extender containing cryoprotectants in order to minimize intracellular crystallization. Glycerol is the most commonly used penetrating cryoprotectant for cryopreservation of mammalian semen and effectively preserves post-thaw sperm function (motility, viability and acrosome integrity) [3]. Despite its benefits, some studies have reported low fertility rates when boar spermatozoa were frozen with extenders containing a high concentration of glycerol (4–8%) [4–8]. Recently, several amides, such as dimethylacetamide (DMA) and dimethylformamide (DMF) have been used as an effective substitute for glycerol in the freezing protocols for boar, stallion, rooster and rabbit semen [7,9–11]. Since most amides are hydrophilic by nature and have a lower molecular weight compared to glycerol, they may induce a limited osmotic damage to spermatozoa [12]. In addition, diverse chemical structures and hydrophilic nature of cryoprotectants might trigger diverse reactions with the sperm cells that could affect post-thaw semen quality [13–16]. The freezing and thawing rates also influence sperm cryosurvival [17,18]. An optimal cooling rate must therefore be determined to minimize cryoinjuries, which include the disruption of the sperm plasma membrane and DNA structure, induced by extensive intracellular ice formation as well as changes in the intracellular pH and ionic composition [17,19,20]. Our previous study demonstrated that favorable straw freezing methods for glycerol-based extenders were different from those with DMA, and the interaction that occurred between the cooling velocity and cryoprotectants affected post-thaw boar semen quality [8]. Furthermore, the experimental design of the current study was principally derived from our previous research [8], which showed that the addition of Equex STM paste to extenders containing 5% DMA or 5% glycerol could improve the cryosurvival of boar spermatozoa, and highlighted the beneficial effects of the straw freezing method with different cryoprotectants. However, there were low fertility rates in preliminary artificial insemination (AI) trials using extenders containing 5% DMA or 5% glycerol. Therefore, the aim of the current study was to evaluate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and DMA (0%, 1%, 3%, and 5%) as well as the straw freezing method (liquid nitrogen or dry ice) on post-thaw boar semen quality.
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Introduction
2.
Materials and methods
2.1.
Semen collections
The gel-free fraction of boar ejaculate (approximately 200– 350 mL) was collected into a pre-warmed plastic container
(38 8C) covered with gauze, using the gloved-hand technique. The ejaculates were collected from two or three Duroc boars, giving a total of seven ejaculates. Sperm motility was evaluated subjectively with a phase contrast optical microscope (Olympus BX50, Tokyo, Japan) at 100 magnification. Only ejaculates with ≥70% motility were processed. Sperm concentration was calculated, using a haemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) [21].
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2.2.
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Semen cryopreservation
Immediately after collection, the ejaculates were diluted (1:1; v/v) in Beltsville Thawing Solution extender (BTS; Minitüb, Abfüll-und Labortechnik GmbH & Co. KG, Tiefenbach, Germany) [22] and transferred to the laboratory in a container (16 8C) within 60 min. On arrival at the laboratory, each extended semen sample was immediately centrifuged at 800 T g for 10 min at 16 8C. The supernatant was discarded and the sperm pellets were re-suspended (1:1; v/v) in a lactoseegg yolk extender (11% lactose solution (w/v) and 20% egg yolk, 0.5% Equex STM paste). Subsequently, each semen sample was divided in 32 equal parts for the treatment, stored in a container for 60 min at 16 8C and then further cooled for 120 min at 5 8C. Following cooling at 5 8C, the freezing extender containing glycerol (Sigma, St. Louis, MO, USA) and/or N,Ndimethylacetamide (DMA, Sigma) was added to the samples. The freezing extenders consisted of different concentrations of glycerol (0, 1, 2, 3 or 5%) and/or DMA (0, 1, 3 or 5%) yielding 16 samples to be frozen in dry ice (D-treatments) and 16 samples to be frozen in liquid nitrogen (N-treatments). The specific concentrations of glycerol and DMA in each D- or N-treatment are depicted in first three columns of Tables 2 and 3. Semen sample was diluted (2:1; v/v) in the freezing extender and packaged in 0.5 mL straws (400–450 106 spermatozoa/mL). The first set of cooled 16 semen samples (D samples) was frozen by placing straws horizontally at the surface of dry ice for 10 min, whereas the second set of 16 samples (N samples) were frozen by placing the straws horizontally at 3 cm above the surface of liquid nitrogen for 20 min. At the end, all frozen straws were stored in liquid nitrogen until required. For sperm assessments, the frozen straws were thawed for 20 s at 37 8C, and the semen samples were re-suspended (1:20, v/v) in a prewarmed (37 8C) BTS extender.
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2.3.
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Semen evaluation
Ten microliters of diluted semen were placed on a microscopic slide, covered with a cover glass (18 mm 18 mm) and at least 10 microscopic fields were examined at 200 magnification under a phase contrast optical microscope (Olympus BX50) equipped with a pre-warmed (38 8C) stage. Sperm viability and morphology were studied using nigrosin and eosin [23]. Morphological abnormalities of spermatozoa were classified as major or minor [24]. Only major abnormalities were recorded when both major (e.g., teratoid head, kinked midpiece, double tails) and minor abnormalities (e.g., detached head, simple bent or coiled tail, terminally coiled tail) simultaneously occurred in a spermatozoon. Acrosome integrity of fixed spermatozoa was assessed using a fluorescein iosthiocyanate labeled peanut agglutinin
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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(FITC-PNA; E.-Y. Lab. Inc., San Mateo, CA, USA) [25] with some modifications [26]. Briefly, frozen-thawed spermatozoa were washed by centrifugation (600 g, 5 min) with phosphatebuffered saline (PBS) to remove non-sperm components and fixed in 0.5% glutaraldehyde and 2% paraformaldehyde (Sigma). After washing, the fixed spermatozoa were stained with 20 mg/mL FITC-PNA for 30 min at 37 8C. The washing procedure was repeated to remove the remaining FITC-PNA and subsequently counter-stained with propidium iodide (FertiLight® kit, Molecular Probes Inc. Eugene, OR, USA) for 10 min at 37 8C. Spermatozoa exhibited two major fluorescent patterns when observed under epifluorescence microscope (Nikon OPTIPHOT-2, Tokyo, Japan): (1) spermatozoa with intact acrosome lacked green fluorescence over the apical portion of the head region and equatorial segment, and (2) spermatozoa with damaged acrosome exhibited a distinct green fluorescence over the apical portion of the head region or equatorial segment. The acrosome integrity was assessed in at least 200 spermatozoa per sample. Sperm longevity was determined by calculating the proportions of motile and viable spermatozoa following a 6-h post-thaw incubation period at 37 8C.
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2.4.
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Fourteen multiparous sows were used for the AI experiment. Each sow was checked for heat twice a day. All sows were artificially inseminated at 24, 36 and 48 h after standing heat, and semen was delivered into cervix with a catheter [21]. The insemination was carried out by the same operator. Three and four sows were separately inseminated with post-thaw semen of 9D-treatment and 10N-treatment, respectively, whereas seven sows were inseminated with fresh semen diluted in BTS. The farrowing rate (%) and total number of piglets were recorded.
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2.5.
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Post-thaw sperm quality was analyzed in duplicate. The statistical analysis was performed using SPSS (SPSS, V12.0, SPSS Inc., Chicago, IL, USA). Before the analysis, normal distribution of data was confirmed. The percentage data were analyzed by generalized linear model (GLM) and incorporated an ANOVA factorial design to study the main effects of cryoprotectant (at various concentrations of glycerol and DMA), freezing method (dry ice and liquid nitrogen) and incubation time (0 and 6 h; only sperm motility and viability) and their interactions on the
Artificial insemination (AI)
Statistical analysis
analyzed post-thaw sperm parameters. If ANOVA showed significant differences among treatment means, a planned multiple comparison of means was examined by post hoc Tukey's test. Values are presented as the mean SEM, and differences were considered significant at p < 0.05.
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3.
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Results
The mean values of sperm motility, viability and acrosome integrity for fresh semen extended in BTS were 86%, 88% and 91%, respectively. The ANOVA results of the three main effects (cryoprotectant, freezing method and incubation time) for post-thaw semen quality are shown in Table 1. Cryoprotectant significantly affected post-thaw semen quality except for normal sperm morphology. Freezing method significantly influenced post-thaw sperm acrosome integrity and normal morphology, whereas incubation time had a significant effect on motility and viability of frozen-thawed spermatozoa. Among the sixteen D-treatments, the best scores for postthaw semen quality were observed in the 16D group (5% glycerol alone), with sperm motility, viability and acrosome integrity determined as 48 3%, 53 3%, and 52 1%, respectively (Table 2). There were no significant differences (p > 0.05) in post-thaw semen quality between the 16D group and the 9D (3% glycerol alone) group regardless of the incubation time. In addition to 16D and 9D groups, the 5D-, 10D-, 11D- and 15D-treatments also had beneficial effects on post-thaw semen quality in terms of sperm motility and viability (Table 2). Among the sixteen N-treatments, the best scores for postthaw semen quality were observed in the 10N group (3% glycerol, 1% DMA), with sperm motility, viability and acrosome integrity determined as 44 3%, 48 3%, and 45 1%, respectively (Table 3). There were no significant differences (p > 0.05) observed between the 10N group and 9N (3% glycerol alone) group regardless of the incubation time. Post-thaw sperm morphology did not differ (p > 0.05) among the examined groups either within the D-treatment (Table 2) or the N-treatment (Table 3). Preliminary AI trials showed that the farrowing rate and litter size obtained with using fresh semen were 86% and 10.2, respectively. The farrowing rates and litter sizes obtained in the 9D group and 10N group were 33% and 6, and 50% and 6.5, respectively.
Table 1 – ANOVA sources of variations in post-thaw boar semen quality. Sperm parameters Sources Cryoprotectant (C) Freezing method (F) Incubation time (I) CF CI FI
Motility
Viability
Acrosome integrity
Normal morphology
<0.001 >0.177 <0.001 <0.001 <0.001 >0.514
<0.001 >0.150 <0.001 <0.001 <0.001 >0.490
<0.001 <0.003 – <0.001 – –
>0.147 <0.012 – >0.137 – –
Significant at p < 0.05.
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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Table 2 – Effects of various concentrations of glycerol and/or DMA on post-thaw boar semen quality (mean W SEM) following freezing in dry ice. Treatment
Cryoprotectant Gly %
1D 2D 3D 4D 5D 6D 7D 8D 9D 10D 11D 12D 13D 14D 15D 16D
1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 5
Sperm parameters (%)
DMA %
0 1 3 5 0 1 3 5 0 1 3 5 1 3 5 0
0h
6h
Motility
Viability
Acrosome integrity
Normal morphology
Motility
Viability
26 2 cd 28 2 cd 33 3bcd 22 2 d 38 3 abc 34 3bcd 33 2bcd 24 2 d 47 4 a 42 2ab 38 1abc 32 2bcd 23 2 d 23 3 d 40 2ab 48 3a
28 3 cd 30 2 cd 35 3 bcd 23 2 d 41 4 abc 36 3 bcd 35 2 bcd 26 2 d 50 4 a 45 2 ab 41 1 abc 34 3 bcd 24 2 d 25 3 d 43 2 ab 53 3 a
29 1 fg 30 1 f 36 1 e 26 1 h 41 1 cd 38 1 de 37 1 e 28 1 fgh 50 1 a 46 1 b 40 1 cd 35 1 e 26 1 gh 27 1 gh 42 1 c 52 1 a
89 1 88 2 88 1 90 1 86 2 88 3 87 2 89 2 87 1 89 1 88 2 87 3 89 2 88 2 89 1 88 1
8 1 cd 11 1 bcd 9 1 cd 7 1 cd 12 1 abcd 13 3 abcd 9 1 cd 8 1 cd 18 3 ab 15 1 abc 10 1 cd 6 1d 6 1d 9 1 cd 11 3 abcd 19 2 a
9 1 cd 11 1 bcd 9 1 cd 8 1 cd 13 1 abcd 13 4 abcd 9 2 cd 9 1 cd 20 3 ab 16 1 abc 10 1 cd 7 1d 7 1d 10 1 cd 12 4 bcd 21 2 a
D – dry ice; Gly – glycerol; DMA – dimethylacetamide. Within a column, values with different letters differ significantly ( p < 0.05). n = 7 ejaculates.
Table 3 – Effects of various concentrations of glycerol and/or DMA on post-thaw boar semen quality (mean W SEM) following freezing in liquid nitrogen. Treatment
Cryoprotectant Gly %
1N 2N 3N 4N 5N 6N 7N 8N 9N 10N 11N 12N 13N 14N 15N 16N
1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 5
Sperm parameters (%)
DMA %
0 1 3 5 0 1 3 5 0 1 3 5 1 3 5 0
0h
6h
Motility
Viability
Acrosome integrity
Normal morphology
Motility
Viability
33 2abcde 22 1 e 28 2 cde 30 2 cde 35 2 abcd 34 3 abcde 28 3 cde 24 2 de 43 2 ab 44 3 a 33 1 abcde 31 3 bcde 32 3 abcde 28 2 cde 34 3 abcde 37 4 abc
35 2 abcde 23 1 e 30 2 cde 32 3 cde 38 2 abcd 37 3 abcde 29 3 cde 25 2 de 45 2 ab 48 3 a 35 2 abcde 34 3 bcde 34 3 abcde 29 3 cde 36 4 abcde 40 4 abc
38 1 bcd 28 1 h 33 1 fg 36 1 cde 40 1 b 38 1 bc 33 1 efg 28 1 h 44 1 a 45 1 a 32 1 fg 35 1 cdef 35 1 def 31 1 g 35 1 def 40 1 b
86 2 88 2 86 2 87 3 85 1 87 3 88 2 88 2 89 1 87 1 85 2 88 1 88 3 89 2 87 2 86 2
7 1 de 7 1 de 8 1 cde 6 1e 13 2 abcd 11 1 bcde 7 1 de 6 1 de 16 2 ab 19 1 a 9 1 cde 6 1 de 10 2 bcde 9 1 cde 14 2 abc 17 3 ab
8 1 de 7 1 de 8 1 cde 6 1e 14 2 abcd 12 1 bcde 8 1 de 7 1 de 18 3 ab 20 1 a 10 1 cde 7 1 ef 11 2 de 9 1 bcde 15 2 cde 18 3 abc
N – liquid nitrogen; Gly – glycerol; DMA – dimethylacetamide. Within a column, values with different letters differ significantly (p < 0.05). n = 7 ejaculates.
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The effects of two cryoprotectants with two straw freezing methods on post-thaw boar semen quality were examined in the current study. The results clearly demonstrated that the semen sample with extender containing 5% glycerol and
Discussion
frozen in dry ice (16D-treatment) yielded the highest mean values of post-thaw sperm motility, viability, and acrosome integrity compared with other treatments. Some reports have demonstrated low fertility rates when boar spermatozoa were frozen in extenders containing a high concentration of glycerol (4–8%) [4–6]. Corcuera et al. [6] reported that spermatozoa frozen in high concentrations of glycerol might compromise
Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008
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the sperm acrosome integrity and increase chromatin condensation and stability. These increases, in turn, may induce early embryonic death or lower embryonic development [27]. Although we did not observed the detrimental effect of high glycerol concentrations on post-thaw sperm acrosome integrity in the current or previous studies [8]. However, low farrowing rates (0–8.3%) were evident in our preliminary AI trials with post-thawed semen frozen in extender containing 5% DMA or 5% glycerol (unpublished data). Interestingly, in the present study, the post-thaw semen qualities of extenders with 3% glycerol alone were similar to those with 5% glycerol alone (9D-treatment vs. 16D-treatment or 9N-treatment vs. 16N-treatment). However, this phenomenon was not observed when semen was preserved in extenders with DMA alone (14D-treatment vs. 15D-treatment or 14N-treatment vs. 15N-treatment). In the present study, the experimental procedure involved a modified protocol from that used in a previous study [8]. Unpublished data and previous research indicated that the cytotoxicity of high glycerol concentrations (4–8%) on fertility could be decreased by modifying the cryopreservation process (e.g., altering the freezing method and lowering cryoprotectant concentrations), and those modifications could further improve post-thaw sperm quality, such as fertilizing potential [4–8]. In the present study, we evaluated the fertilizing ability of frozen-thawed spermatozoa using a glycerol-based extender (9D-treatment) and extender containing both glycerol and DMA (10Ntreatment). Only 7 sows were used in AIs owing to financial limitations; however, the 9N-treatment and other treatments that had beneficial effects on sperm cryosurvival will be considered for further AI trials. In the present study, we also evaluated whether the combinations of different concentrations of two penetrating cryoprotectants (glycerol and DMA) could achieve similar or improve the cryosurvival of boar spermatozoa [4–6]. Other than the combinations of 1% glycerol and 1 or 3% DMA for samples frozen in dry ice (2D- or 3D-treatment), or 3% glycerol and 1% DMA for samples frozen in liquid nitrogen (10Ntreatment), DMA had no beneficial effects on sperm cryosurvival when added to the glycerol-based extender. This possibly indicates that the combination of glycerol and DMA did not significantly affect the cryosurvival of boar spermatozoa. Similar results were observed by Malo et al. [28], who demonstrated that DMF was less effective than glycerol in the cryopreservation of boar semen, when used alone or in combination with glycerol. Kim et al. [29] showed that DMA and dimethylsulfoxide (DMSO) were less effective than glycerol in improving post-thaw semen quality. Furthermore, Moustacas et al. [30] demonstrated that DMF had no beneficial effects on post-thaw quality of ram semen when used alone or in combination with glycerol. It should be emphasized that the penetrating cryoprotectants have unique structures and characteristics that could influence the mechanism of interaction with cellular structures, ultimately affecting sperm cryosurvival [15]. The results of the present study indicate that DMA might interfere with the interaction of glycerol and the sperm structure, intracellular ice formation, and osmolarity, which could induce increased cytotoxicity in sperm function. The present study compared the effects of two simple freezing methods on post-thaw semen quality. The results
5
indicated that the straw-freezing method using dry ice was more favorable for the extender containing 5% glycerol or 5% DMA (15D- vs. 15N-treatment or 16D- vs. 16N-treatment). However, Wu et al. [8] demonstrated that the straw-freezing method using liquid nitrogen was more favorable for extenders containing 5% DMA. The major difference between the results was the higher sperm concentration used in the present study (800–900 106 sperm/mL) than in the previous study (150 106 sperm/mL). Although glycerol and DMA, as penetrating cryoprotectants, allow samples to be frozen immediately without incubation [6,8], high sperm concentrations may hinder the interaction of glycerol or DMA with the sperm structures. This could affect the rate of osmoticallydriven water flow, which may affect membrane permeability, intracellular pH, and ionic composition [17,19,20]. Therefore, prolonged incubation and slower freezing rates might have contributed to the beneficial effects in the present study and other studies [31]. Corcuera et al. [6] observed improved motility and acrosome integrity when boar spermatozoa were incubated in 2% glycerol-based extender for 30 min at 5 8C before cryopreservation. Since the freezing rate differed among treatments due to different spermatozoa and cryoprotectant concentrations or exposure time, a controlled-rate freezer was used to establish the optimal freezing rate, as discussed by Thurston et al. [32]. In conclusion, the results of the present study indicated that a combination of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa. However, the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the most effectual post-thaw quality parameters of boar semen.
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Conflict of interest
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All authors declare that they have no conflict of interests.
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Please cite this article in press as: Yang C-H, et al. Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality. Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2015.12.008