Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders

Theriogenology 66 (2006) 2047–2055 www.journals.elsevierhealth.com/periodicals/the

Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders Ana Martins-Bessa a,*, Anto´nio Rocha b, A. Mayenco-Aguirre c a

Department of Veterinary Sciences and CECAV, UTAD, Quinta de Prados, 5001-801 Vila Real, Portugal b ICBAS and ICETA/CECA, University of Porto, Oporto, Portugal c Department of Medicine and Animal Surgery, Faculty of Veterinary, UCM, Madrid, Spain Received 23 January 2006; received in revised form 14 June 2006; accepted 14 June 2006

Abstract The objective of this work was to evaluate the possibility of substituting glycerol (G) for ethylene glycol (EG) when cryopreserving dog semen. A total of 15 ejaculates from 13 dogs was pooled into five samples and frozen in egg-yolk Tris extenders with variable ethylene glycol and glycerol concentrations, with or without Equex1 STM Paste. Two widely used glycerol extenders (Uppsala Equex II and Norwegian) were utilized as controls. Semen quality parameters assessed after thawing were total subjective motility (TSM), computer assisted sperm analysis (CASA), eosin-nigrosin staining, and flow cytometry (FC) after staining with the PI/Fitc-PSA (fluorescein isotiocianate conjugated with the agglutinin of Pisum sativum, PSA) fluorochromes. No advantages were seen in using EG to replace G when freezing dog semen or combining EG and G in the freezing medium. The Uppsala Equex II provided the best overall post-thaw parameters, followed by the egg-yolk Tris experimental extender with 5% EG and Equex1 STM Paste. The extender with 4% EG produced similar results to the Norwegian extender. High correlations (r > 0.98) were obtained between eosin-nigrosin staining and FC, as well as between subjective and computerized motility assessment (r > 0.90). # 2006 Elsevier Inc. All rights reserved. Keywords: Dog; Frozen semen; Glycerol; Ethylene glycol; Equex1 STM Paste

1. Introduction Glycerol (G) is the most commonly used cryoprotectant for dog sperm. After the first artificial insemination of dogs with semen frozen using a Tris extender with 8% glycerol [1], several other extenders with variable glycerol concentrations have been tested. The results of these studies [2–6] varied by extender and utilized a wide range (2–8%) of glycerol concentrations. Discrepancies between studies as to the ideal glycerol concentration

* Corresponding author. Tel.: +351 259350639; fax: +351 259350480. E-mail address: [email protected] (A. Martins-Bessa). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.06.004

could be due to several factors such as using different extenders, using different cryopreservation protocols, using other cryoprotectants (such as Equex STM Paste) and using different criteria/methods to evaluate postthaw sperm quality. Conversely, glycerol, besides its cryoprotective properties, can induce alterations in the organization and viscosity of the sperm cytoplasm and in the permeability and stability of the plasma membrane through disruption of phospholipid and protein structural organization [7–9]. These phenomena can cause potential deleterious effects on the sperm fertilizing ability. Recently, the incorporation of Equex STM Paste in freezing extenders for dog sperm was shown to have a beneficial effect in post-thaw motility and integrity of the membrane [10–12], which could enhance the attachment

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of the sperm cell to the zona pellucida [13]. This paste is composed of sodium-dodecyl-sulphate, a biological detergent that exerts a protective action. This action seems to be related to the interaction between the eggyolk and the sperm membrane, thereby increasing sperm permeability and reducing osmotic stress during the freezing and thawing processes [14]. Despite the success of freezing canine sperm using glycerol as a cryoprotectant, other cryoprotectants such as dimethylsulfoxide (DMSO) and ethylene glycol (EG) have been tried. The incorporation of DMSO, an intracellular cryoprotectant, did not improve post-thaw motility and survival rates of frozen-thawed dog semen [2] or ram semen [15]. EG has successfully been used to freeze mouse and chinchilla sperm [16,17]. Results of previous work in dogs comparing EG and G as a sperm cryoprotectant are still few and the results contradictory. Cavalcanti et al. [18] reported lower post-thaw motility with EG than with G when the same concentration was used, whereas Soares et al. [19] observed similar postthaw motility with EG at 0.25, 0.5 and 1 M as with the glycerol at 0.8 M. Santos et al. [20] obtained similar results for motility, vigor and morphology preservation of thawed semen, using either EG or G at 5%. More recently, Rota et al. [21] using 5% EG or 5% G with Equex STM Paste saw a positive effect of EG at thawing in both motility and membrane integrity, as determined by the hypoosmotic swelling test (HOS). The chemical structures of EG and G are quite similar, both having the same ratio of carbon atoms and C/OH hydroxyl groups, an indicator of the molecule lipophilia/ hydrophilia [22]. However, EG has a smaller (62.07 versus 92.10) molecular weight, a characteristic that may result in lower toxicity and higher permeability to cells [23]. Consequently, EG is widely used for embryo freezing in various mammalian species [23– 25], as well as freezing of ovarian tissue [26,27]. The effects of using EG in frozen/thawed semen varied among species. With bull semen, EG resulted in higher post-thaw motility when compared with G or DMSO. This may be because of a reduction of the ‘‘osmotic lesions’’ [28]. The possibility that EG could cause less ‘‘osmotic lesions’’ had already been suggested for stallion spermatozoa [29]. When used to cryopreserve stallion semen, EG had results similar to those of glycerol, and successfully replaced it when used in the same or lower concentrations [30,31]. This experiment was designed to determine the feasibility of using different concentrations of EG as a cryoprotectant for canine semen, alternatively or combined with G or with Equex STM Paste1. Our hypothesis was that EG would improve post-thaw motility, viability

and integrity of the acrosome and plasma membrane. The experimental extenders were compared with the Norwegian [1] and the Uppsala Equex II [32] extenders that are widely used for clinical and research purposes. 2. Materials and methods 2.1. Animals Semen from thirteen dogs, two to five years old, of different breeds (seven German Shepherd Dogs, two Rottweilers, three Labrador Retrievers and one Cocker Spaniel) kept in the kennel of the Policia Nacional of Madrid was collected. Two dogs were collected twice. The dogs were clinically healthy and had proven fertility after natural mating. 2.2. Extenders Extenders composed of TRIS-citric acid-egg-yolk and variable ethylene glycol concentrations with or without Equex STM Paste, as well as a glycerol-EG combination were used. Percentages of EG in the TRIS extender were: 4% EG (extender EG4), 8% EG (extender EG8), 4%EG and 4% G (extender EG4G4), and 5% EG with 0.5% Equex STM Paste (extender EG5Eq). The Norwegian (NWG: 8% glycerol) [1] and Uppsala Equex II (UEqII: 5% glycerol with 0.5% Equex STM Paste) [32] extenders were used as controls. The composition of the control extenders was: UEqII component A: 3.025 g TRIS1, 1.7 g citric acid monohydrate2, 1.25 g fructose3, distilled water to 77 mL, 3 mL glycerol4, 0.06 g benzyl-penicillin5, 0.1 g streptomycin6, 20 mL egg-yolk, pH 6.74, 988 mOsm; UEqII component B: 3.025 g TRIS, 1.7 g citric acid monohydrate, 1.25 g fructose, distilled water to 72 mL, 7 mL glycerol, 1 mL Equex STM Paste7; 0.06 g benzylpenicillin, 0.1 g streptomycin, 20 mL egg-yolk, pH 6.74, 2905 mOsm [32]; NWG: 6.05 g TRIS, 3.4 g citric acid monohydrate, 2.5 g fructose, 16 mL glycerol, 0.12 g benzylpenicillin, 0.2 g streptomycin, 40 mL eggyolk, distilled water to 200 mL, pH 6.62, 331 mOsm [1]. The composition of the experimental extenders was: EG4: 6.05 g TRIS, 3.4 g citric acid monohydrate,

1 2 3 4 5 6 7

Panreac 141940, Madrid, Spain. Panreac 141018. Panreac 142728. Panreac 141339. Sigma P-3032, St. Louis, MO, USA. Sigma S-9137. Nova Chemical Sales Inc., Scituate, MA, USA.

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2.5 g fructose, 8 mL ethylene glycol8, 0.12 g of benzylpenicillin, 0.2 g streptomycin, 40 mL egg-yolk, distilled water to 200 mL, pH 6.74, 1368 mOsm; EG8: 6.05 g TRIS, 3.4 g citric acid monohydrate, 2.5 g fructose, 16 mL ethylene glycol, 0.12 g of benzyl-penicillin, 0.2 g streptomycin, 40 mL egg-yolk, distilled water to 200 mL, pH 6.74, 2807 mOsm; EG4-G4: 6.05 g TRIS, 3.4 g citric acid monohydrate, 2.5 g fructose, 8 mL glycerol, 8 mL ethylene glycol, 0.12 g of benzylpenicillin, 0.2 g streptomycin, 40 mL egg-yolk, distilled water to 200 mL, pH 6.73, 2993 mOsm; EG5Eq: component A: 3.025 g TRIS, 1.7 g citric acid monohydrate, 1.25 g fructose, distilled water to 77 mL, 3 mL ethylene glycol, 0.06 g benzyl-penicillin, 0.1 g streptomycin, 20 mL egg-yolk, pH 6.74, 1058 mOsm; component B: 3.025 g TRIS, 1.7 g citric acid monohydrate, 1.25 g fructose, distilled water to 72 mL, 7 mL ethylene glycol, 1 mL Equex STM Paste; 0.06 g benzylpenicillin, 0.1 g streptomycin, 20 mL egg-yolk, pH 6.74, 2961 mOsm. The extenders were prepared a week in advance, divided into aliquots and frozen at 20 8C until used. 2.3. Semen collection and evaluation, dilution and freezing The sperm-rich fraction of the ejaculate from each day was collected into a calibrated plastic tube, by digital manipulation. Motility was estimated immediately after collection, using a microscope9 at 400 magnification, equipped with a heated stage kept at 38 8C. Only ejaculates with at least 75% estimated progressive motility, 75% normal morphology and with 250  106 total spermatozoa were used. Semen smears were made for morphology assessment with Spermac1 stain (Stain Enterprises, Republic of South Africa). After individual examination, the ejaculates were pooled together (three ejaculates for each of the five pools) and the motility of the pool was evaluated. The pool was then divided into six aliquots and centrifuged at 700  g for 5 min at room temperature. Each aliquot was diluted with one of the six extenders to a final concentration of 50  106 spermatozoa/mL and the extended semen was allowed to equilibrate for 2 h in a refrigerator set at 4 8C. If the extender contained Equex STM Paste (UEqII and EG5Eq extenders), the aliquot was diluted in component A and allowed to equilibrate

8

Panreac 141316. Olympus CHK3-F-GS Microscope (Olympus Optical Company Limited, Taiwan). 9

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for 60–75 min to 4 8C, and component B was added after equilibration [12]. The chilled semen was then packaged in 0.5 mL IMV1 straws and frozen horizontally 4 cm above the surface of liquid nitrogen for 10 min, before being immersed into the liquid nitrogen. Semen straws were kept frozen in liquid nitrogen for at least one week, before being thawed for evaluation. A total of five pools (three ejaculates/pool) were utilized for analysis. 2.4. Thawing and evaluation Thawing was performed at 70 8C for 8 s. The samples then were diluted 1:1 (v/v) in the Uppsala Equex II thaw medium [32] at 38 8C to a final concentration of 25  106 spermatozoa/mL. Samples were kept in a water-bath at 38 8C in tubes protected from light. Two straws from each extender and from each of the five pools were thawed for subjective (light microscopy) and computerized motility evaluation, and two other were used for longevity assessments. Three straws were thawed for evaluation of plasma and acrosome membrane integrity and two more for evaluation of live/ dead cells after eosin-nigrosin staining. 2.5. Motility and longevity Aliquots of diluted semen were evaluated for subjective and computerized evaluation of motility with the Sperm Class Analyzer (Microptic SL1, Barcelona, Spain) after 3 min of incubation (at 38 8C) in the postthaw medium and at 30 min intervals thereafter, until motility neared 0%. The Sperm Class Analyzer consisted of a phase-contrast Olympus BX50 microscope equipped with a heating plate set at 38 8C, a Sony Video Camera B/ N and the computerized program Sperm Class Analyzer (SCA) 2002 System. The following settings were used in the analyzer: 25 frames acquired per second; minimum area of particles of 20 mm; minimum percentage of straightness for progressive spermatozoa 80; connectivity of 12 and a minimum number of 10 sperm cells to calculate amplitude of lateral head (ALH) displacement. The limit of velocity for immobile objects (mm/s) was adjusted at each assessment, in such a way that spermatozoa without movement were classified as static. The persistence of motility in thawed semen diluted in each medium was designated as ‘‘Longevity’’. Six to 10 sequences of each sample were captured and recorded in the SCA for further analysis. In each sequence the following characteristics of 150–200 spermatozoa were analyzed: % total motility, TM; % progressive motility,

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PM; curvilinear velocity (mm/s), VCL; rectilinear velocity (mm/s), VSL, average path velocity (mm/s), VAP; linearity (%), LIN and amplitude of lateral head displacement (mm), ALH. Total subjective motility (TSM) was visually assessed using a phase contrast microscope at 200  magnification. The correlation between subjective (TSM) and computerized (TM) motility was evaluated in all 30 samples at thawing, at 30 min and at 1 h post-thawing, in 29 samples at 1 h 30 min postthawing, in 26 samples at 2 h post-thawing and in 19 samples at 2 h 30 min post-thawing. The number of samples decreased over time due to some batches displaying motility near 0%.

were excited by an Argon laser at a wave length of 488 nm. A minimum of 20,000 sperm cells was counted in the FACScan Cytometer (Becton Dickinson, San Jose, CA, USA) for presence of fluorescence caused by incorporation of PI, Fitc-PSA and their combination with PI. Data was analyzed using the software Cell Quest (Becton Dickinson, San Jose, CA, USA) and expressed as percentage of cells that emitted fluorescence. For eosin-nigrosin staining, a drop of approximately 10 mL of semen was gently mixed with the same volume of the stain on a warm slide. Correlations between FC analysis (PI ) and eosin-nigrosin were assessed for 30 samples, for times T0 and T1:30 postthaw.

2.6. Integrity of the acrosome and sperm plasma membranes

2.7. Statistical analysis

The simultaneous evaluation of the integrity of the acrosome and sperm plasma membranes was performed by flow cytometry (FC) after staining with PI10FitcPSA11 (fluorescein isotiocianate conjugated with the agglutinin of Pisum sativum, PSA). The stock-solution was diluted to a concentration of 0.1 mg/mL (work solution). Controls were systematically carried out for each extender and fluorochrome, using live and dead sperm cells as well as a mixture of live and dead samples. Semen samples were processed using the techniques described in detail by Pen˜a et al. [33] and Go´mez-Cue´tara [34]. Briefly, a 400 mL aliquot of thawed semen was filtered through a 34 mm nylon mesh (distributed by Nessler SA, Madrid, Spain) and rediluted in PBS to a final concentration of 10  106 sperm cells/mL. A sample of 40 mL was taken from this aliquot, and 3 mL of PI (0.1 mg/mL) were added and incubated for 5 min at room temperature, after which 5 mL of Fitc-PSA (0.1 mg/mL) was also added. The sample was then filtered in the nylon mesh and analyzed by FC. Analyses were also carried out in all the samples after 1 h 30 min of incubation at 38 8C, always protecting the thawed semen from light. The association PI/Fitc-PSA allows the detection of four sperm sub-populations by FC [33]: (1) live sperm (intact plasma membrane) with intact acrosome (PI PSA ); (2) live sperm with damaged or reacted acrosome (PI PSA+); (3) dead sperm with intact acrosome (PI + PSA ); and (4) dead sperm with damaged or reacted acrosome (PI + PSA+). Samples

10 11

Sigma P-4170. Sigma L-0770.

Analysis of variance (ANOVA) was used to compare the different extenders for all the parameters and the Duncan test was applied for multiple comparisons of means among groups. Statistical analyses were done on data collected until 2 h 30 min post-thaw. After that, the reduced number of viable samples for some extenders prevented further analysis. Correlation between subjective (TSM) and computerized (TM) motility, and FC analysis (PI ) and eosin-nigrosin assessment for live sperm were analyzed with Pearson’s correlation coefficients. All statistical analyses were performed with the SPSS 11.0 programme [35]. Differences were considered significant for p < 0.05. Data are presented as mean  standard deviation (S.D.). 3. Results 3.1. Fresh semen Mean (S.D.) fresh semen parameters (n = 15) were 85.83%  4.1% TM, 75.83%  3.76% PM, 89.10%  3.5% of cells with normal morphology and a concentration of 379.27  168.11  106 sperm cells/mL. 3.2. Post-thaw sperm characteristics 3.2.1. Motility, acrosome and plasma membrane integrity and longevity The motility patterns (TM, PM and TSM) were best overall in the UEqII and EG5Eq extenders (Tables 1–3). Although TM, PM and TSM in these two extenders were not different from three of the other extenders (NWG, EG4 and EG4-G4) at time 0 (Tables 1–3), the progressive motility was greater at time 1 for both extenders and at time 1:30 for UEqII (Table 3). At time

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Table 1 Post-thaw total subjective motility (TSM-mean  S.D.) of five pools of canine semen frozen in different extenders (% motile) measured at thawing (TSM 0) and at each 30 min Extender

TSM 0

TSM 0:30

TSM 1

TSM 1:30

TSM 2

TSM 2:30

UEqII EG5Eq NWG EG4 EG4-G4 EG8

73.40  5.50b 73.00  5.70b 73.00  2.74b 66.00  9.62b 65.00  10.00b 53.00  13.96a

69.00  7.42b 67.00  8.37b 63.00  6.71b 60.00  11.18b 45.00  10.61a 39.00  10.84a

66.00  4.18d 63.40  11.84cd 46.00  7.42bc 41.00  21.33ab 31.00  11.94ab 24.00  18.84a

65.00  5.00c 52.00  16.81c 21.00  17.73ab 32.00  18.23b 22.00  7.58ab 9.00  10.83a

59.00  11.40b 27.40  25.13a 14.80  14.69a 12.50  9.57a 10.50  10.21a 6.33  7.50a

47.00  14.83b 26.75  22.11ab 17.33  16.62a 6.68  4.16a 11.00  12.73a 6.00  5.66a

<0.001

<0.001

<0.001

<0.001

p-Value

0.008

0.022

Means in the same column with different superscripts differ significantly. Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4-G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

Table 2 Post-thaw total motility (TM-mean  S.D.) of five pools of canine semen frozen in different extenders (% motile) measured at thawing (TM 0) and at each 30 min Extender UEqII EG5Eq NWG EG4 EG4-G4 EG8 p-Value

TM 0

TM 0:30 b

68.48  9.56 70.02  5.35b 66.08  7.53b 59.10  12.64ab 58.20  18.15ab 44.62  11.65a 0.044

TM 1 c

64.62  19.81 59.64  15.38bc 53.24  12.59bc 50.06  12.74bc 39.66  14.84ab 27.92  13.66a 0.007

TM 1:30 c

TM 2 b

64.96  15.36 60.10  13.19c 37.80  8.36b 34.92  16.73b 28.42  9.56ab 14.70  9.40a

63.96  12.63 52.16  20.60b 21.40  12.69a 27.54  16.07a 19.34  7.93a 10.28  7.61a

<0.001

<0.001

TM 2:30 b

57.66  19.99 28.74  19.55a 17.06  10.68a 14.95  9.42a 16.60  10.98a 11.33  9.78a 0.001

41.08  20.99 29.47  16.60 20.40  9.81 14.30  7.80 16.65  8.41 11.20  7.35 ns

Means in the same column with different superscripts differ significantly. Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4-G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

2 UEqII produced the best results for all the motility parameters (TM, PM and TSM). The linearity for semen frozen with UEqII was also greater than in 4 (NWG, EG4, EG8 and EG4-G4) of the other 5 extenders, but only at times 0:30 and 1 when compared to extenders EG4 and EG8 (Table 4). Semen frozen with EG5Eq extender had inferior results for some post-thaw

parameters (PM at 1:30 h and TM, TSM and PM at 2 h post-thaw) and integrity of the acrosome and sperm plasma membranes parameters (Tables 1–3 and 5) than the UEqII extender. The extenders without Equex STM Paste and using EG as cryoprotectant (EG8 and EG4) did not produce better results than the extender with G (NWG). Using EG and G in the same extender (extender

Table 3 Post-thaw progressive motility (PM-mean  S.D.) of five pools of canine semen frozen in different extenders (% motile) measured at thawing (PM 0) and at each 30 min PM 0 UEqII EG5Eq NWG EG4 EG4-G4 EG8 p-Value

PM 0:30 b

57.00  12.65 55.54  6.13b 57.84  10.65b 49.38  14.5ab 45.02  14.02ab 35.38  14.42a 0.062

PM 1 c

54.88  18.61 47.50  18.27bc 38.62  14.58abc 35.44  14,18abc 27.87  13.88ab 18.98  8.66a 0.012

PM 1:30 c

PM 2 c

56.20  16.02 47.76  15.43c 27.66  9.59b 22.32  13.07ab 19.08  6.04ab 8.34  7.54a

52.44  12.29 36.50  15.38b 10.68  8.52a 14.76  8.75a 11.70  4.98a 7.50  7.33a

<0.001

<0.001

PM 2:30 b

43.44  19.77 18.78  16.89a 8.48  7.94a 8.50  6.25a 6.83  5.44a 4.77  3.57a 0.002

26.72  18.04 26.43  4.05 15.35  5.44 7.90  5.54 10.50  4.80 5.50  6.08 ns

Means in the same column with different superscripts differ significantly. Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4-G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

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Table 4 Post-thaw linearity rate (LIN-mean  S.D.) of five pools of canine semen frozen in different extenders (%) measured at thawing (LIN 0) and at each 30 min LIN 0

LIN 0:30

LIN 1

LIN 1:30

LIN 2

LIN 2:30

UEqII EG5Eq NWG EG4 EG4-G4 EG8

84.42  4.55 78.56  3.86 83.16  4.87 79.56  8.04 80.72  5.54 77.88  3.46

80.54  4.06c 74.86  4.05bc 70.04  4.63ab 67.18  3.46a 73.40  5.78ab 66.94  5.47a

76.40  1.61c 73.82  3.40bc 70.68  7.22bc 68.70  3.43b 73.48  2.79bc 61.42  8.94a

78.66  9.83 65.66  11.74 56.72  20.54 64.42  11.75 67.18  2.71 56.72  17.58

72.28  10.59 56.26  19.33 47.12  22.39 57.43  20.76 55.97  14.30 65.90  8.00

64.12  13.26 55.92  22.26 55.00  30.80 64.43  8.00 66.40  13.29 61.90  6.15

p-Value

ns

ns

ns

ns

0.001

0.003

Means in the same column with different superscripts differ ( p < 0.05). Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4-G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

Table 5 Mean (S.D.) of different sperm sub-populations after staining with fluorochromes PI-Fitc-PSA and FC analysis (%) measured at thawing (T0) and after 1 h 30 min of incubation at 38 8C: live sperm with intact acrosome (PI PSA ); live sperm with damaged or reacted acrosome (PI PSA+); dead sperm with intact acrosome (PI + PSA ) and dead sperm with damaged or reacted acrosome (PI + PSA+) PI

PSA

(%)

T0 UEqII EG5Eq NWG EG4 EG4-G4 EG8 p-Value

T 1:30 b

55.4  10.9 37.8  11.0a 36.5  12.1a 36.1  11.9a 27.5  8.6 a 28.7  14.3a 0.014

43.3  6.6 31.2  9.6 30.3  12.9 28.8  11.9 26.4  7.9 25.9  19.2 ns

PI + PSA+ (%)

PI + PSA

(%)

PI

T0

T0

T 1:30

T0

T 1:30 a

27.0  5.9 37.2  6.3ab 44.8  5.2b 44.7  11.7b 47.2  7.7b 51.6  19.3b 0.018

33.6  5.3 41.2  6.2 42.6  9.1 46.1  10.6 46.3  8.8 53.8  19.4

16.3  4.0 22.2  4.9 15.9  3.5 15.9  4.2 18.6  0.6 13.2  6.4

21.0  3.5 24.7  6.1 22.8  13.4 20.5  3.6 22.7  4.3 14.4  6.8

ns

ns

ns

PSA+ (%)

(%)

T0

T 1:30

1.5  1 2.0  0.1 a 2.8  2.2 ab 3.0  0.8 ab 3.1  1.4 ab 4.7  2,3 b

56.9  10.7 39.9  10.9 38.6  11.9 38.3  13.3 33.2  7.4 35.0  15.6

44.8  6.7 33.3  10.1 33.1  12.5 31.8  11.6 29.5  8.7 30.6  18.7

0.05

ns

ns

T 1:30 a

1.2  0.6 2.0  0.9 a 2.1  1.0 a 2.2  0.8 a 5.7  2.2 b 6.3  1.9 b <0.001

TOTAL PI

a

Means in the same column with different superscripts differ significantly. Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4-G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

Table 6 Mean (S.D.) of five pools for post-thaw longevity measured in minutes, for semen frozen in different extenders Extender

Longevity (min)

UEqII EG5Eq NWG EG4 EG4-G4 EG8

252.00  40.30b 216.00  57.70ab 186.00  65.04ab 168.00  50.20a 168.00  45.50a 144.00  39.12a

p-Value

0.031

Means in the same column with different superscripts differ significantly. Tris-based extenders with egg-yolk: EG4 (4% ethylene glycol); EG5Eq (5% ethylene glycol with 0.5% Equex STM Paste); EG4G4 (4% ethylene glycol with 4% glycerol); NWG (8% glycerol); UEqII (5% glycerol with 0.5% Equex STM Paste); EG8 (8% ethylene glycol).

EG4-G4) resulted in lower ( p < 0.001) TSM at time 0:30 and greater acrosome damage at thawing than the one obtained by all but the EG8 extender (Tables 1 and 5). Semen frozen with EG8 had the lowest TSM at thawing (Table 1). The two control (UEqII and NWG) extenders and the experimental EG5Eq extender resulted in comparable sperm post-thaw longevity (Table 6). Staining with PI-Fitc-PSA allowed for the detection of differences among extenders for percentage of live sperm cells with intact acrosome (PI PSA ) and for percentage of dead sperm cells with damaged acrosome (PI + PSA+) at time 0 and for percentage of reacted live cells (PI PSA+) at time 0 and time 1:30 (Table 5). The UEqII extender had the greatest numbers of live sperm with intact acrosomes (Table 5) and, along with EG5Eq, had the fewest dead sperm with damaged acrosomes. No significant differences among extenders were seen

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for total percentage of sperm cells that did not incorporate PI (total PI ) (Table 5). 3.3. Correlations between analyzed parameters The correlation between TSM and TM was high in all observed time-periods (r = 0.90–0.96; p = 0.01– 0.05). The correlation between FC analysis (PI ) and eosin-nigrosin staining at T0 was 0.992 and 0.986 at T1:30 ( p = 0.01). 4. Discussion The use of EG for cryopreservation of stallion [30,31,36] or ram [15,37] semen can provide similar or better results than those obtained with G. Results of previous work in dogs comparing EG and G as semen cryoprotectant are scarce and contradictory, with ethylene glycol resulting in similar or better preservation in some studies [19–21] and giving inferior results in another [18]. In our study, the best post-thaw motility results were obtained with the UEqII and EG5Eq extenders, the only ones with Equex STM Paste, which suggests a beneficial effect of the paste, as previously noted [10–12]. However, the design of this experiment did not allow objective evaluation of the effect of Equex STM Paste. From the extenders without Equex STM Paste, the ones with EG did not produce better results than the extender with G, similarly to what was previously found by Cavalcanti et al. [18] working with 4% and 7% EG and 7% G. Contrary to these results, a recent experiment found a positive effect of the EG at thawing when compared with G, both at a concentration of 5% [21]. The effects of the EG as a semen cryoprotectant may be species-specific. Some of the disadvantages of EG may be related to toxicity to the sperm cells. In fact, a toxic effect of EG on human sperm has been suggested due to its deleterious effect on the motility of frozen and fresh samples [38]. One could speculate that a toxic effect could be a reason for the poor results obtained in the current study with EG at 8%, as the utilization of a lower concentration (EG at 4%) produced results comparable to those obtained with G at 8%. Similarly, the slightly lower effectiveness of extender EG5Eq compared to UEqII could also be due to a slight toxic effect of the EG. Further studies utilizing lower EG concentrations associated with Equex STM Paste could contribute to clarify this hypothesis. It has been also suggested that EG could not reverse the sperm capacitation-like changes that occur in the freezing process [21].

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The association of EG with G (EG4-G4) did not result in any additional benefits. This absence of a beneficial effect of the EG/G association is in agreement with results obtained with ram semen, where the association resulted in poorer post-thaw motility and acrosomal integrity [15]. The authors of that study attributed the results to a possible EG toxicity. Other differences contributing to the different effects of EG versus G may be the lower molecular weight of EG, which allows it to penetrate and leave the cell faster than other cryoprotectants [19]. Thus, EG could minimize the detrimental effects of the dehydration and re-hydration during the freezing/thawing processes. In fact, equine sperm had a higher osmotic tolerance to quick addition and removal of EG, than to G, DMSO or propylene-glycol [29]. Similarly, the addition and removal of ethylene glycol seemed to minimize alterations of cellular volume of bovine spermatozoa, when compared with using DMSO or G [28]. However, permeability of the sperm cell membrane to either EG or G, and the consequent osmotic-induced alterations of cellular volume, may also be species-specific. In fact, G penetrates dog sperm cells quickly [6], as it does in swine sperm [39] where it reaches equilibrium in less than 30 s. The semen dilution method during equilibration could also have influenced the post-thaw quality. The two step dilution, adding a portion of the cryoprotectant before equilibration and the rest after, seems advantageous over adding all the cryoprotectant before equilibration. This has been shown with the Uppsala Equex method [12,40] and adding all the cryoprotectant after equilibration has been reported in another study [41]. The combination of several in vitro evaluation methods to study different characteristics of the sperm cells increases the accuracy of the evaluation of the spermatic function [42]. Furthermore, the characteristics of sperm motility determined by computerized systems indirectly reflect the integrity of the sperm cell membrane, as well as the cellular metabolic activity [43]. Subjective (visual) evaluation of sperm motility and morphology has been utilized in previous work using EG as a cryoprotectant for freezing dog semen [18–20], and is a practical, quick and easy to use method. However, its subjective nature makes comparing results among different investigators, questionable [9]. In the present work, the subjective evaluation of the motility was complemented with the use of a computerized system SCA1. Differences among extenders were found for total subjective motility, total (TM) and progressive (PM) computerized motility, linearity rate (LIN) and integrity of the membrane by means of staining with PI/Fitc-PSA. The poorer results of the EG8 for some parameters were

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not evident for parameters assessing velocity (VCL, VSL and VAP, data not show). On the other hand, the good maintenance of motility and other positive post-thaw characteristic observed for semen frozen in UEqII were not accompanied by a significant increase of velocity parameters or by a decrease of ALH, contrary to what has been observed by others [12]. This discrepancy could be due, among other factors, to differences in sperm concentrations in the straws utilized in the two studies and to the impact that this factor seems to have on sperm post-thaw quality [44]. Highly significant correlations were found between viability parameters measured by means of PI/FC and by eosin-nigrosin using light microscopy, which is in agreement with previous work [45]. Similarly, highly significant correlations were also found between visual and computerized motility evaluation, as previously noted [46]. These findings indicate that despite the increased information obtained with more recent and sophisticated techniques, some simple and fast methods for evaluating semen viability remain useful. However, it should be taken into account that subjective assessments require considerable experience and expertise. In conclusion, no advantages were seen in using EG to replace G in dog semen freezing. Similarly, no benefits were seen by combining EG and G in the freezing medium. The UEqII extender provided the best post-thaw parameters. From all the experimental extenders (EG4, EG8, EG4-G4, EG5Eq), EG5Eq produced the best results, being superior to the control NWG extender. Finally, some simple methods to assess viability of sperm cells produced results that were comparable to the ones obtained by more objective techniques. Acknowledgements The authors thank the National Police of Madrid (Spain) for allowing semen collection from their dogs. A. Martins-Bessa was supported by a Ph.D. Grant from Fundac¸a˜o para a Cieˆncia e a Tecnologia (BD/SFRH/ 7076, Ministe´rio da Cieˆncia, Tecnologia e Ensino Superior, Portugal), and by European Social Found (European Union). The authors also gratefully acknowledge the critical review and English language corrections done to a previous version of this manuscript, by Dr. Bruce Eilts from Louisiana State University. References [1] Andersen K. Insemination with frozen dog semen based on a new insemination technique. Zuchthygiene 1975;10:1–4.

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