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Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen
Journal of Equine Veterinary Science 33 (2013) 1068-1075
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen Diégo Moreno DMV, Djemil Bencharif DMV, PhD, Lamia Amirat-Briand DMV, PhD, Alberto Neira DMV, PhD, Sandrine Destrumelle DMV, PhD, Daniel Tainturier DMV, PhD Laboratory of Biotechnology and Pathology of Reproduction, Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering, Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2012 Received in revised form 26 February 2013 Accepted 3 April 2013 Available online 15 May 2013
The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at þ37 C, centrifuged (600 g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 106 spermatozoa/ml. The resulting mixture was cooled to 4 C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at þ4 C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at 196 C and stored. Two straws per extender and per ejaculate were thawed in a water bath at þ37 C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at þ37 C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: LDL Stallion Spermatozoa Cryopreservation
Corresponding author at: Djemil Bencharif, DMV, PhD, Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: Ecole Nationale Vétérinaire, Agroalimentaire et d’Alimentation, BP 40706, 44307 Nantes, France. E-mail address: [email protected] (D. Bencharif). 0737-0806/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2013.04.004
1. Introduction Artificial insemination is used extensively in animal reproduction. Freezing semen enables commercialization
D. Moreno et al. / Journal of Equine Veterinary Science 33 (2013) 1068-1075
of the sperm of stud animals with a high genetic value on a global scale. However, the freezing-thawing process causes irreversible damage to the cytoplasmic membrane of the spermatozoa, possibly due to the formation of ice crystals inside the cells, which leads to the death of the gamete. Semen extenders with cryoprotective substances are therefore required. Stallion semen presents numerous problems during cryopreservation. After thawing, the fecundity of the spermatozoa decreases [1]; this phenomenon is a function of the individual and the ejaculate. Thus, one in two stallions has semen that is unsuitable for freezing or of very poor quality. In one study of freezability of stallion semen at the French National Stud between 1985 and 2005, 5%, 4%, 5%, 21%, and 64% of the stallions had a semen freezability of 010%, 10-33%, 33-60%, 60-90%, and over 90%, respectively [28]. Numerous semen extenders have been studied to try to overcome this problem, and particular attention has been paid to cryoprotectants. Currently, three substances are regularly combined for equine semen: milk, egg yolk, and glycerol. Egg yolk is a component of most semen extenders, because it protects the sperm from thermal shock during freezing [9,29]. Despite the protective effect of egg yolk, a few of its components exert a noxious effect on spermatozoa [23]. Kampshmidt et al. [15] demonstrated that the granules found in egg yolk can reduce the respiration and motility of spermatozoa in the bull. Furthermore, egg yolk can be a source of bacterial contamination [26]. The cryoprotective fraction of egg yolk, represented by low-density lipoprotein (LDL) was therefore isolated and purified [7,8,12,17,21,23]. LDLs are the principal components in egg yolk, representing 2/3 of the dry matter of the yolk. They are primarily located in the plasma, but a small portion is found in the granules. LDLs are spherical particles between 17 and 60 nm in diameter, with a nucleus of neutral lipids (triglycerides and cholesterol) surrounded by a monofilm of phospholipids and proteins (apoproteins). Phospholipids have an essential role in the stabilization of the structure of LDL, due to the hydrophobic character of their bonds [11]. Cholesterol is included in the phospholipid film, increasing its rigidity. LDLs consist of 83%-89% lipids and 11%-17% proteins, with a pH between 6.3 and 7.5 [22]. The lipids are composed of 74% neutral lipids and 26% phospholipids [18]. LDLs are the principal emulsifying agents of egg yolk; this ability is clearly due to their structure via interactions between the apoproteins and the phospholipids [5]. The use of LDL (purified) from egg yolk in semen extenders instead of whole egg yolk improves the postthaw motility rate of spermatozoa in the bull [4,21,27], dog [6], billy goat [3], and boar [14]. The beneficial effects of egg yolk plasma (LDL, high-density lipoprotein [HDL], phosvitins, and livetins) in a freezing extender were recently demonstrated in the stallion by Pillet et al. [24]. Egg yolk plasma gave results similar to that of egg yolk in terms of motility, membrane integrity, and fertility. However, the effects of the purified LDL fraction obtained from egg yolk plasma in an extender for freezing stallion semen have not been studied, and the appropriate concentration of LDL in semen extenders, which varies from one species to another, has yet to be determined.
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The aim of this study was to verify the cryoprotective effect of LDL (purified) in an extender for freezing equine semen. The first step was to determine the appropriate concentration of LDL to obtain the best post-thaw motility results, evaluated using an image analyzer. 2. Materials and Methods 2.1. Animals The study included 14 ejaculates collected from four stallions of different breeds: Arabian, pony, and Selle Français, with an average age of 8 years, 3 of which were privately owned and 1 that belonged to the National Veterinary, Agrofood, and Food Hygiene School of Loire Atlantique (ONIRIS), Nantes, France. Samples were taken using a closed artificial Missouri vagina (IMV Technologies, L’Aigle, France) filled with water at þ42 C and lubricated with petroleum jelly. The samples were taken using a dummy mount or with a teaser mare next to the dummy. After collection, the sperm was filtered through sterile gauze to eliminate the gel fraction. The volume of the semen was then recorded, the spermatozoal concentration was determined using a photometer (Sperma Cue; Minitube, Germany), and spermatozoal motility was analyzed using computer-assisted semen analysis (CASA). Ejaculates with a concentration of more than 60 106 spermatozoa/ml and a motility of more than 50% [25] were used for this study. 2.2. Extenders 2.2.1. Extraction of LDL The LDLs were extracted using the technique developed by Moussa et al. [21]. Fresh hen eggs were manually broken. Yolks were separated from the albumen and carefully rolled on filter paper to remove chalazas and traces of albumen adhering to the vitelline membrane. The latter was pierced with a scalpel blade, and yolk was collected into a beaker placed on ice. Yolks were diluted with an isotonic saline solution (0.17 M NaCl; w/w) and stirred for 1 hour before being centrifuged at 10,000 g for 45 minutes at 4 C. The supernatant (plasma) was separated from the sediment (granules). Plasma was centrifuged again to remove all traces of contamination with granules; it was then mixed with 40% ammonium sulfate to precipitate out livetins. After 1 hour of stirring at 4 C, the mixture was centrifuged at 10,000 g for 45 minutes. The sediment was discarded and the supernatant dialyzed against distilled water to eliminate the ammonium sulfate. Finally, the solution was centrifuged (10,000 g, 45 minutes, 4 C) and the LDL-rich supernatant was collected. 2.2.2. Preparation of the Extenders We prepared 10 different extenders: INRA 82 with 2% egg yolk (control), INRA 96 (control; IMV Technologies, L’Aigle, France), and INRA 82 with 8 different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), used in centrifugation of the semen. A second series of the same 10 extenders was then prepared but with the addition of 2.5% glycerol (Table 1); these were used in the freezing of the semen.
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Table 1 Composition of different extenders for the centrifugation and freezing of equine sperm, with and without glycerol Composition
LDL 0.25%
General composition
Glucose 2.5 g; lactose 0.15 g; raffinose 0.15 g; sodium citrate 0.025 g; potassium citrate 0.041 g, Hepes 0.476 g; penicillinestreptomycin 0.5 ml; distilled water 50 ml; skimmed milk 50 ml. e e e e e e e e 2 0.680 1.360 2.041 2.721 5.44 8.165 10.88 13.6 e 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 335 334 334 336 335 334 335 338 368
e e 100 ml 301
2.5 100 ml 626
2.5 100 ml 651
Egg yolk (ml) LDL (g) Total volume (QSP) Osmotic pressure without glycerol (mOsm/kg) Glycerol (ml) Total volume (QSP) Osmotic pressure with glycerol (mOsm/kg)
LDL 0.50%
2.5 100 ml 624
LDL 0.75%
2.5 100 ml 627
LDL 1%
2.5 100 ml 626
LDL 2%
2.5 100 ml 626
LDL 3%
2.5 100 ml 627
LDL 4%
2.5 100 ml 628
LDL 5%
2.5 100 ml 628
2% EY Control
2.5 100 ml 764
INRA96 Control e
EY, egg yolk; LDL, low-density lipoprotein; QSP, qunatity sufficient for.
2.3. Freezing The ejaculate was divided into 10 tubes. The sperm in each tube was diluted into equal parts (v/v) at þ37 C with each prepared extender but without glycerol. The tubes were centrifuged at 600 g for 10 minutes to eliminate seminal plasma. The pellet was put back into suspension in the 10 freezing extenders (those containing glycerol) to obtain a final dilution of 100 106 spermatozoa/ml. After dilution, the sperm was cooled from þ37 C to þ4 C in a refrigerated cabinet at þ4 C for 60 minutes. The sperm was then packed into 0.5-ml straws (IMV Technologies), four straws for each extender tested, and sealed with polyvinyl alcohol. The straws were left on a metal ramp for 30 minutes at þ4 C. After equilibration, the straws were frozen by placing them in the vapors 4 cm above liquid nitrogen (120 C) for 10 minutes. Finally, they were immersed in the liquid nitrogen (196 C).
(VSL: mm/s), average path velocity (VAP: mm/s), and amplitude of the lateral head displacement (ALH: mm/s).
2.6. Assessment of Spermatozoal Integrity The last two straws of each extender tested were thawed in a water bath at þ37 C for 30 seconds. Four tests were conducted.
2.5. Motility Analysis
2.6.1. Assessment of Plasma Membrane Integrity: Hypo-Osmotic Test The integrity of the plasma membrane was assessed using the hypo-osmotic (HOS) test to observe the transport of liquids across the plasma membrane [13]. For this test, 25 ml of sperm was diluted in 25 ml of a hypo-osmotic solution (100 mOsm/kg H2O) prepared with 75 mM fructose and 25 mM tri-sodium-citrate in distilled water. After incubation at þ37 C for 60 minutes, a smear was made with 5 ml of the mixture. The slides were then observed under the microscope (CK2, ULWCD 0.30; Olympus) at a magnification of 400 (200 spermatozoa were observed). Spermatozoa were classified as positive or negative depending on the presence or absence, respectively, of a curled flagellum.
Semen was analyzed twice: before freezing (after incubation for 60 minutes at þ4 C in the different extenders) and after thawing (after incubation at 37 C for 10 minutes). An analyzer (Hamilton Thorne) with Ceros 12 software (IMV Technologies, Aigle, France) was used. After thawing, 2 ml of semen were sampled using a micrometric pipette and placed in the chamber of a Leja cell (CryoBio system; IMV Technologies). To prevent thermal shock, the cells were initially placed on a heated stage at þ37 C and observed at a magnification of 200. Five fields were chosen at random over the chamber and analyzed, a mean was then taken. Twenty images per second were taken. The objects present in the cell were recognized as being spermatozoa as a function of their size and brightness. The following parameters were analyzed: motility (% motile spermatozoa), progressively motile spermatozoa (% progressive sperm cells: average path velocity higher than 50 mm$s1, and straightness of track higher than 70%), curvilinear line velocity (VCL: mm/s), straight line velocity
2.6.2. Assessment of Acrosome Integrity: Pisum sativum Agglutinin-Fluorescein Isothiocyanate Test The integrity of the acrosome was assessed using the Pisum sativum agglutinin-fluorescein isothiocyanate (PSAFITC) test, in accordance with the technique described by Mendoza et al. [19]; 5 ml of the sperm being studied was used to make one smear. After being air dried, the slide was immersed in a solution of 100% methanol for 15 minutes to fix and permeabilize the membrane. After drying, the slide was incubated with 200 ml of a solution of PSA-FITC (50 mg/ml) for 10 minutes at room temperature in the dark. The slide was then rinsed and immersed in distilled water for 15 minutes and then air dried at þ4 C. The evaluation was conducted with fluorescence microscopy (DM-IRB model; Leica Microsystems, Wetzlar, Germany). Two hundred spermatozoa were observed. The positive spermatozoa presented with selective staining of the acrosome (intact spermatozoa), while the negative sperm did not take up the stain.
2.4. Thawing Thawing was achieved by immersing two straws of each extender in a water bath at þ37 C for 30 seconds [25].
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2.6.3. Evaluation of DNA Structure: Acridine-Orange Test The structure of the chromatin was assessed using the acridine-orange (AO) stain test. For this test, 150 ml of the sperm was diluted with 4 ml of PBS that had been incubated in a water bath at þ37 C. The tubes were centrifuged at 500 g for 10 minutes. Then, 100 ml of the pellet was mixed with 100 ml of the TNE buffer solution (0.01 M TrisHCl, 0.15 M NaCl, and 1 mM disodium EDTA, pH ¼ 7.4). The suspension of sperm was then mixed with 400 ml of Triton solution (0.1% Triton, 0.15 M NaCl, 0.08 M HCl, pH ¼ 1.2). After 30 seconds of incubation, 1.2 ml of acridine orange (0.2 M Na2HPO4, 1 mM disodium EDTA, 0.15 M NaCl, 0.1 M citric acid, and 6 ml/ml acridine orange, pH ¼ 6.0) were added to the mixture, followed by incubation in the dark for 5 minutes to enable fixation of the AO. Finally, 5 ml of suspension was spread on a slide and covered with a coverslip. The slides were observed with fluorescence microscopy (DM-IRB; Leica) at a wavelength of 520 nm. For each slide, 200 spermatozoa were counted and classified into two groups according to the color of their head: green staining indicated the presence of native (double-stranded) DNA, and orange staining indicated the presence of denatured (single-stranded) DNA. 2.6.4. Evaluation of Spermatozoal Morphology: Spermac Stain Spermatozoal morphology was assessed using the Spermac stain (Minitube). The reagents were placed in 20ml flasks. A smear was made with the semen to be evaluated and left to air dry for 5 minutes. The slide was then fixed by immersion in the flask containing the fixative for 5 minutes. After being air dried for 1 hour, the slide was rinsed five to six times with distilled water, and excess water was removed with absorbent paper. The slide was then plunged into reagent A (red) to be stained for 2 minutes and rinsed with distilled water. A second staining was performed for 1 minute in reagent B (pale green). After rinsing, a third and final staining was performed in a bath containing reagent C (green) for 1 minute before being rinsed again. The slides were observed under the microscope with oil-immersion objective. The acrosome stains dark green, the nucleus red, the equatorial region pale green, and the intermediate section of the tail green.
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Table 2 Motile and progressive spermatozoa in the various extenders Extender
M (%)
0.25% LDL 0.50% LDL 0.75% LDL 1% LDL 2% LDL 3% LDL 4% LDL 5% LDL Egg yolk (control) INRA 96 (control)
76.6 79.7 78.7 78.5 80.4 80.7 78.5 76.8 76.6 72.4
PS (%) 2.1ab 1.9 2.3 2.6 2.1 2.3 2.6b 2.4ab 3.1ab 2.7ab
39.2 41.6 40.8 41.0 43.0 42.3 40.7 39.5 38.8 39.4
1.7ab 1.8 2.1a 2.2a 2.7 2.3 2.1ab 2.0ab 2.4ab 2.1
Table shows mean (SEM) percentages of motile (M) and progressive spermatozoa (PS) in the various extenders at T0 þ 60 minutes, at þ4 C before freezing (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
Two hundred spermatozoa were counted and classified as normal or abnormal. The various types of anomalies were classified according to their location, that is, anomalies of the spermatozoa head, vesicles, damaged acrosome, double heads, and decapitated heads and anomalies of the intermediate piece (swollen) and anomalies of the flagellum (swollen flagellum or cut flagellum). 2.7. Statistical Analysis An analysis of variance (ANOVA) model was used with repeated values according to a linear model with a mixed effect, studied using S-plus statistical software, to determine the influence of the various treatments on motility and progressive spermatozoa, as well as the various motility parameters; these same tests were also applied to the results of the various stains. The equality of variances test using paired results enables a comparison extender by extender. 3. Results 3.1. Motility and Percentage of Progressive Spermatozoa At time 0 (T0), immediately after sperm collection, and before dilution in the various extenders, the mean SD
Fig. 1. Comparison of percentages of motile and progressive spermatozoa. Values are means (SEM). Comparison of the percentages of motile and progressive spermatozoa in various extenders at T0þ10 minutes after thawing (n ¼ 14). (a) P < .05 versus 2% LDL; (b) P < .05 versus 3% LDL.
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motility was approximately 88.7% 6.29% with an average of 42.7% 9.27% progressive spermatozoa for the 14 ejaculates collected. The mean percent of motility before freezing (incubation at þ4 C for 1 hour) was statistically higher (P < .05) with the extenders made with 2% and 3% LDL than with the control extenders made with egg yolk and INRA 96 and with the 0.25% and 5% LDL extenders. There were no significant differences with the 0.50%, 0.75%, and 1% LDL extenders (Table 2). For the progressive spermatozoa, the 2% LDL extender was significantly superior (P < .05) to the control extender made with egg yolk and to the 0.25%, 0.75%, 1%, 4%, and 5% LDL extenders, but there were no significant differences with the INRA 96 extender and the 0.5% and 3% LDL extenders (Table 2). The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant differences were observed among these extenders. The 2% and 3% LDL extenders were significantly superior (P < .05) to those obtained with the control INRA 96 and to the 0.25%, 1%, and 5% LDL (Fig. 1). The post-thaw progressive spermatozoa percentages with the 0.50%, 0.75%, 2%, and 3% LDL extenders and the extender containing egg yolk were significantly superior (P < .05) to those obtained in the 0.25% and 5% LDL and the INRA 96 extenders (Fig. 1). 3.2. Study of Movement Characteristics of Spermatozoa Before freezing (incubation at þ4 C for 1 hour), the extenders containing 0.25% and 0.50% LDL gave superior results for VAP, VSL, and VCL. There was a significant difference (P < .05) between these extenders and all of the other extenders in the study. There were no significant differences among the ALH values in all of the extenders (P > .05) (Table 3). After thawing, the best VAP, VSL, and VCL values were obtained with the extenders containing low concentrations of LDL (0.25%, 0.50%, and 0.75%) and the control extender containing egg yolk. These extenders were significantly superior (P < .05) to the other concentrations of LDL and to the INRA96 extender. Again, there were no significant differences (P > .05) in ALH values among any of the extenders (Table 3). 3.3. Assessment of Spermatozoal Integrity 3.3.1. HOS Test Membrane integrity with the extenders containing 0.50%, 0.75%, 1, 2, 3 and 4% LDL were significantly superior (P < .05) to those obtained with the control extender INRA 96, but no significant difference was observed with the control egg yolk extender. The 2 and 3% LDL extenders were significantly superior (P < .05) to those obtained with the 0.25, 1, and 5% LDL (Table 4). 3.3.2. PSA-FITC Test The percentage of spermatozoa with green stained acrosomes (intact) was approximately 83% for all of the extenders. The high concentrations of LDL (4% and 5% LDL) gave percentages significantly lower than those of the 0.5%, 1%, 3% LDL and INRA96 extenders (P < .05). However, no
Table 3 Automated motility parameters before freezing and after thawing Group
VAP (mm/s)
VSL (mm/s)
Before freezing (T0 þ 60 min at 4 C) 0.25% LDL 90.6 1.2ab 67.0 0.6ab 0.50% LDL 90.5 1.6ab 66.1 1.4ab 0.75% LDL 88.0 1.7b 65.0 1.3b 1% LDL 86.7 2.1 64.1 1.6 2% LDL 86.0 1.7 64.0 1.6 3% LDL 85.8 1.7 63.4 1.4 4% LDL 85.2 1.8 63.2 1.4 5% LDL 84.6 1.9 62.7 1.2 Egg yolk 87.8 1.4a 64.6 1.2 (control) INRA 96 76.6 2.1ab 57.6 1.5ab (control) After thawing (T0 þ 10 min at 37 C) 0.25% LDL 69.8 3.1ab 59.0 2.6ab 0.50% LDL 68.7 2.9ab 58.0 2.4ab 0.75% LDL 69.9 2.3ab 59.8 1.9ab 1% LDL 66.3 3.1 55.8 2.7 2% LDL 65.6 2.9 55.5 2.4 3% LDL 64.6 2.3 55.0 1.7 4% LDL 65.5 1.9 56.3 1.4 5% LDL 61.7 2.4ab 52.6 1.8b Egg yolk 70.8 2.6ab 59.8 2.1ab (control) INRA 96 53.6 2.0ab 45.4 1.9ab (control)
VCL (mm/s)
153.8 154.9 152.0 152.0 149.7 150.0 148.1 146.4 152.0
2.7ab 2.4ab 2.2 2.3 2.1 2.3 2.5 3.5 1.7
141.5 3.3ab
115.8 115.4 115.0 112.1 110.2 109.0 108.6 104.2 117.0
4.3ab 4.4ab 3.2ab 4.5 3.8 4.1 3.4 3.9ab 3.8ab
95.6 3.6ab
ALH (mm/s) 5.7 5.9 5.8 6.0 5.9 5.9 5.8 5.7 5.9
0.12 0.11 0.10 0.11 0.13 0.14 0.14 0.15 0.10
6.0 0.14
4.9 4.8 4.6 4.9 4.7 4.5 4.5 4.6 4.8
0.17 0.17 0.16 0.17 0.18 0.20 0.18 0.14 0.20
4.8 0.13
ALH, amplitude of lateral head displacement; VAP, average path velocity; VCL, curvilinear line velocity; VSL, straight line velocity. Mean (SEM) automated motility parameters before freezing and after thawing (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
significant differences were observed between the different concentrations of LDL and the control egg yolk extender (P > .05) (Table 5). 3.3.3. AO Test According to the equality of variances on paired observations test, there was no significant difference between the different extenders with the AO test. DNA was preserved in 98% of the spermatozoa in all of the extenders. 3.3.4. Spermac Stain After thawing, over 70% of the spermatozoa did not present any anomalies; 78% for the extenders containing 0.25% and 2% LDL and the control extender containing egg yolk (Fig. 2). All of the values were similar, except for the 5% LDL extender, which gave a percentage that was significantly lower (P < .05) than that of the other extenders. The
Table 4 Percentages of intact plasma membranes detected by the HOS test after thawing as a function of the extenders LDL LDL LDL LDL 0.25% 0.50% 0.75 1% SEM 27.0ab 31.3 SEM 1.7 1.9
LDL LDL LDL LDL 2% 3% 4% 5%
EY 2%
INRA 96
29.5 28.9ab 32.3 32.4 29.7 26.5ab 31.3 23.3ab 2.1 1.5 1.2 1.7 2.1 1.8 1.5 1.4
Mean (SEM) percentages of intact plasma membranes detected by the HOS test after thawing, as a function of the extenders (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
D. Moreno et al. / Journal of Equine Veterinary Science 33 (2013) 1068-1075 Table 5 Percentages of spermatozoa with an intact acrosome (PSA-FITC test) after freezing-thawing in the various extenders LDL LDL LDL LDL 0.25% 0.50% 0.75% 1% SEM 84.3 SEM 1.0
85.8a 85.0 1.4 1.1
LDL 2%
LDL 3%
LDL 4%
LDL 5%
EY 2%
INRA 96
84.5 84.0 84.7 83.2b 82.2b 83.5 84.8 0.8 1.2 0.8 0.9 1.3 1.3 1.3
Mean (SEM) percentages of spermatozoa with an intact acrosome according to PSA-FITC test results after freezing-thawing in the various extenders (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
most common types of anomalies in all the extenders were those of the flagellum, followed by the head and intermediate piece. 4. Discussion The results of this study show that LDL at a concentration of 2% and 3% significantly improve spermatozoal motility and the percentage of progressive spermatozoa following the chilling of equine semen in comparison with the extender containing egg yolk and INRA 96 supplemented with 2.5% glycerol after 1 hour at þ4 C (P < .05). After the freeze-thaw process, the rates were also superior, but not significantly so, than those obtained with the egg yolk extender. With the higher concentrations of LDL (4% and 5%), the plasma membrane and acrosome integrity were less well preserved, and the percentage of spermatozoa with anomalies increased. This confirms previous findings where the best results for the cryopreservation of stallion sperm were achieved with 2% egg yolk, whereas in bulls and dogs, a concentration of 20% was needed. With LDL, the best results for freezing were obtained with a concentration of 8% in the bull [4] and 6% in the dog [6]. In the horse, LDLs have a cryoprotective effect but at a concentration between 2% and 3%. The exact concentration could be determined with a higher number of stallions, possibly 2.5%.
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Khlifaoui [16] has already shown the beneficial effect of LDL for the cryopreservation of equine sperm but at a concentration of 0.5%. For most stallions in this study, the best results were obtained with 2% or 3% LDL; however, in 1 stallion, the 0.5% concentration gave the best results. At the time of sampling, spermatozoal motility was very good, at approximately 90%. The effect of the LDL concentration of the extender could be linked to the composition of the seminal plasma of each stallion, which in turn affects the percentage of motile spermatozoa; this hypothesis needs further investigation. 4.1. Assessment of the Integrity of the Plasma Membrane: HOS Test Spermatozoa with a swollen tail (flagellum) have an intact plasma membrane. Results show that all of the extenders used provided good protection of the flagella plasma membrane, with the exception of INRA 96 supplemented with glycerol. The extenders containing 2% and 3% LDL provided the same level of protection as the egg yolk extender. Because of the low quantity of LDL to be added to the various freezing extenders, the highest percentage of recovered spermatozoa after freezing and thawing was obtained with the 2% LDL extender; while the control extender made with 2% egg yolk has an equivalent LDL concentration of 0.66% LDL. 4.2. Assessment of the Integrity of the Acrosome: PSA-FITC Test It has been proposed that egg yolk contains progesterone [10], which is responsible for the capacitation of spermatozoa and is therefore prejudicial to the preservation of spermatozoa during freezing. This test is justified by the fact that mammalian spermatozoa are incapable of fertilizing the oocyte immediately after ejaculation. It is the fixation of the spermatozoon to the zona pellucida that triggers the acrosome reaction,
Fig. 2. Evaluation of spermatozoal morphology. Values are means (SEM). Comparison of the percentage of spermatozoa without anomalies (Specmac test) in the various extenders after thawing (n ¼ 14). (a) P < .05 versus 2% LDL; (b) P < .05 versus 3% LDL.
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making it capable of fertilization. Spermatozoa that spontaneously undergo an acrosome reaction after ejaculation or following freezing are incapable of fixing to the zona pellucida and are therefore unable to fertilize the oocyte [32]. The extenders containing 2% or 3% LDL provided good protection of acrosome integrity, possibly via a direct action through the exchange or repair of acrosomal membrane phospholipids or possibly simply because the extender has a lower progesterone content than egg yolk because of the filtering effect of the dialysis membrane. The progesterone found in egg yolk plays a role in the capacitation of spermatozoa in cattle [31], horses [2], and humans [20,30]. It appears to act via an extragenomic action on human spermatozoa, via the secondary activation of calcium channels leading to an increase in intracellular Ca2þ, which may be responsible for the capacitation of spermatozoa [2]. Such a mechanism has also been demonstrated in the dog [31]. 4.3. Assessment of DNA Integrity: Acridine Orange Test The integrity of the DNA or chromatin is an important factor when assessing spermatozoa fertility. For this study, AO proved to be effective despite the application of fluorescence microscopy. The results obtained are very good, greater than or equal to 98%. This suggests that LDL provides good protection for cellular DNA during the freeze-thaw process of equine semen. Therefore, extenders containing 2% or 3% LDL are good equine semen preservation agents in comparison with extenders containing egg yolk and INRA 96 supplemented with glycerol. This result was found in all analyses performed and concurs with research undertaken in bulls and dogs [4,6,21]. 4.4. Assessment of Acrosome Integrity: Spermac Test After thawing, 78% of the spermatozoa did not present with any abnormalities with the 0.25% and 2% LDL and egg yolk extenders. The 2% LDL extender seems to provide spermatozoal protection during the freeze-thaw process similar to the EY extender. 5. Conclusions This study demonstrated that extenders containing 2% and 3% LDL give percentages of motile spermatozoa during chilling that are significantly superior to those of the current standard extenders made with egg yolk and INRA 96. After thawing, LDL has a very good cryoprotective effect; at a concentration of 2%, it shows cryoprotective qualities similar to the current extender containing egg yolk and results that are superior to the INRA96 extender and other concentrations of LDL. Acknowledgments The authors thank Myriam Larrat, Sylvie Anezo, and Gérard Chatagnon from the Department of Biotechnology of Reproduction Nantes for technical assistance.
References [1] Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci 1987;7:145-73. [2] Aitken RJ, McLaughlin EA. Molecular mechanisms of sperm capacitation: progesterone-induced secondary calcium oscillations reflect the attainment of a capacitated state. Soc Reprod Fertil Suppl 2007;63:273-93. [3] Ali Al Ahmad MZ, Chatagnon G, Amirat-Briand L, Moussa M, Tainturier D, Anton M, et al. Use of glutamine and low density lipoproteins isolated from egg yolk to improve buck semen freezing. Reprod Dom Anim 2008;43(4):429-36. [4] Amirat L, Tainturier D, Jeanneu L, Gérard O, Courtens JL, Thorin C, et al. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender. Theriogenology 2004;61:895-907. [5] Anton M, Huopalahti R, Lopez-Fandiño R, Schade R. Low-density lipoproteins (LDL) or lipovitellenin fraction. In: Anton M, editor. Bioactive egg compounds. Berlin: Springer-Verlag; 2007. [6] Bencharif D, Amirat L, Anton M, Schmitt E, Desherces S, Delhomme G, et al. The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen. Theriogenology 2008;70(9):1478-88. [7] Bergeron A, Crete MH, Brindle Y, Manjunath P. Low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod 2004;70:708-17. [8] Bergeron A, Manjunath P. New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Mol Reprod Dev 2006;73:1338-44. [9] Bogart R, Mayer D. The effects of egg yolk on the various physical and chemical factors detrimental to spermatozoa viability. J Anim Sci 1950;9:143-52. [10] Bowden RM, Ewert MA, Lipar JL, Nelson CE. Concentrations of steroid hormones in layers and biopsies of chelonian egg yolks. Gen Comp Endocrinol 2001;121(1):95-103. [11] Burley RW. Recent advances in the chemistry of egg yolk. CSIRO Food Research Quaterly 1975;35:1-5. [12] Foulkes JA. The separation of lipoprotein from egg yolk and their effect on the motility and integrity of bovine spermatozoa. J Reprod Fertil 1977;49:277-84. [13] Jeyendran R, Van der Ven H, Pérez-Peláez M, Grabo B, Zaneveld L. Development of an essay to asses the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984;70:219-28. [14] Jiang ZL, Li QW, Li WY, Hu JH, Zhao HW, Zhang SS. Effect of low density lipoprotein on DNA integrity of freezingethawing boar sperm by neutral comet assay. Anim Reprod Sci 2007; 99:401-7. [15] Kampshmidt RF, Mayer DT, Herman HA. Lipid and lipoprotein constituents of egg yolk in the resistance and storage of bull spermatozoa. J Dairy Sci 1953;36:733-42. [16] Khlifaoui M. Equine semen freezing: three cryoprotectants interest: glutamine, low density lipoproteins from chicken egg yolk and carnitine. PhD Thesis. University of Rennes. 2004. [17] Manjunath P, Nauc V, Bergeron A, Menard M. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen’s egg yolk. Biol Reprod 2002;67:1250-8. [18] Martin WG, Augustyniak J, Cook WH. Fractionation and characterization of the low-density lipoproteins of hen’s egg yolk. Biochim Biophys 1964;84:714-20. [19] Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 1992;95:755-63. [20] Meyers SA, Overstreet JW, Liu IKM, Drobnis EZ. Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males. J Androl 1995;16:47-54. [21] Moussa M, Martinet V, Trimeche A, Tainturier D, Anton M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozenethawed bull semen. Theriogenology 2002;57:1695-706. [22] Nakamura R, Hayakawa M, Sato Y. Isolation and fractionation of the protein moiety of egg yolk low density lipoprotein. Poultry Sci 1977;56:1148-52. [23] Pace MM, Graham EF. Components in egg yolk which protect bovine spermatozoa during freezing. J Anim Sci 1974;39:1144-9.
D. Moreno et al. / Journal of Equine Veterinary Science 33 (2013) 1068-1075 [24] Pillet E, Labbé C, Batellier F, Duchamp G, Beaumal V, Anton M, et al. Egg yolk plasma can replace egg yolk in stallion freezing extenders. Theriogenology 2011;75:105-14. [25] Samper JC, Morris CA. Current methods for stallion semen cryopreservation: a survey. Theriogenology 1998;49:895-903. [26] Van Wagtendonk-de Leeuw AM, Haring RM, Kaal-Lansbergen LMTE, Den Daas JHG. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract. Theriogenology 2000;54:57-67. [27] Vera-Muñoz O, Amirat-Briand L, Diaz T, Vásquez L, Schmidt E, Desherces S, et al. Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: comparison to Triladyl and Bioxcell. Theriogenology 2009;71:895-900.
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[28] Vidament M. French fields results (1985e2005) on factors affecting fertility of frozen stallion semen. Anim Reprod Sci 2005;89:115-36. [29] Wall RJ, Foote RH. Fertility of bull sperm frozen and stored in clarified egg yolk-Tris-glycerol extender. J Dairy Sci 1999;82:817-21. [30] Wistrom CA, Meizel S. Evidence suggesting involvement of a unique human sperm steroid receptor/Cl[] channel complex in the progesterone-initiated acrosome reaction. Dev Biol 1993;159: 679-90. [31] Witte TS, Schäfer-Somi S. Involvement of cholesterol, calcium and progesterone in the induction of capacitation and acrosome reaction of mammalian spermatozoa. Anim Reprod Sci 2007;102:181-93. [32] Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil JD, editors. The physiology of reproduction. New York: Raven Press; 1994. p. 189-317.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen Diégo Moreno DMV, Djemil Bencharif DMV, PhD, Lamia Amirat-Briand DMV, PhD, Alberto Neira DMV, PhD, Sandrine Destrumelle DMV, PhD, Daniel Tainturier DMV, PhD Laboratory of Biotechnology and Pathology of Reproduction, Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering, Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2012 Received in revised form 26 February 2013 Accepted 3 April 2013 Available online 15 May 2013
The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at þ37 C, centrifuged (600 g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 106 spermatozoa/ml. The resulting mixture was cooled to 4 C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at þ4 C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at 196 C and stored. Two straws per extender and per ejaculate were thawed in a water bath at þ37 C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at þ37 C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: LDL Stallion Spermatozoa Cryopreservation
Corresponding author at: Djemil Bencharif, DMV, PhD, Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: Ecole Nationale Vétérinaire, Agroalimentaire et d’Alimentation, BP 40706, 44307 Nantes, France. E-mail address: [email protected] (D. Bencharif). 0737-0806/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2013.04.004
1. Introduction Artificial insemination is used extensively in animal reproduction. Freezing semen enables commercialization
D. Moreno et al. / Journal of Equine Veterinary Science 33 (2013) 1068-1075
of the sperm of stud animals with a high genetic value on a global scale. However, the freezing-thawing process causes irreversible damage to the cytoplasmic membrane of the spermatozoa, possibly due to the formation of ice crystals inside the cells, which leads to the death of the gamete. Semen extenders with cryoprotective substances are therefore required. Stallion semen presents numerous problems during cryopreservation. After thawing, the fecundity of the spermatozoa decreases [1]; this phenomenon is a function of the individual and the ejaculate. Thus, one in two stallions has semen that is unsuitable for freezing or of very poor quality. In one study of freezability of stallion semen at the French National Stud between 1985 and 2005, 5%, 4%, 5%, 21%, and 64% of the stallions had a semen freezability of 010%, 10-33%, 33-60%, 60-90%, and over 90%, respectively [28]. Numerous semen extenders have been studied to try to overcome this problem, and particular attention has been paid to cryoprotectants. Currently, three substances are regularly combined for equine semen: milk, egg yolk, and glycerol. Egg yolk is a component of most semen extenders, because it protects the sperm from thermal shock during freezing [9,29]. Despite the protective effect of egg yolk, a few of its components exert a noxious effect on spermatozoa [23]. Kampshmidt et al. [15] demonstrated that the granules found in egg yolk can reduce the respiration and motility of spermatozoa in the bull. Furthermore, egg yolk can be a source of bacterial contamination [26]. The cryoprotective fraction of egg yolk, represented by low-density lipoprotein (LDL) was therefore isolated and purified [7,8,12,17,21,23]. LDLs are the principal components in egg yolk, representing 2/3 of the dry matter of the yolk. They are primarily located in the plasma, but a small portion is found in the granules. LDLs are spherical particles between 17 and 60 nm in diameter, with a nucleus of neutral lipids (triglycerides and cholesterol) surrounded by a monofilm of phospholipids and proteins (apoproteins). Phospholipids have an essential role in the stabilization of the structure of LDL, due to the hydrophobic character of their bonds [11]. Cholesterol is included in the phospholipid film, increasing its rigidity. LDLs consist of 83%-89% lipids and 11%-17% proteins, with a pH between 6.3 and 7.5 [22]. The lipids are composed of 74% neutral lipids and 26% phospholipids [18]. LDLs are the principal emulsifying agents of egg yolk; this ability is clearly due to their structure via interactions between the apoproteins and the phospholipids [5]. The use of LDL (purified) from egg yolk in semen extenders instead of whole egg yolk improves the postthaw motility rate of spermatozoa in the bull [4,21,27], dog [6], billy goat [3], and boar [14]. The beneficial effects of egg yolk plasma (LDL, high-density lipoprotein [HDL], phosvitins, and livetins) in a freezing extender were recently demonstrated in the stallion by Pillet et al. [24]. Egg yolk plasma gave results similar to that of egg yolk in terms of motility, membrane integrity, and fertility. However, the effects of the purified LDL fraction obtained from egg yolk plasma in an extender for freezing stallion semen have not been studied, and the appropriate concentration of LDL in semen extenders, which varies from one species to another, has yet to be determined.
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The aim of this study was to verify the cryoprotective effect of LDL (purified) in an extender for freezing equine semen. The first step was to determine the appropriate concentration of LDL to obtain the best post-thaw motility results, evaluated using an image analyzer. 2. Materials and Methods 2.1. Animals The study included 14 ejaculates collected from four stallions of different breeds: Arabian, pony, and Selle Français, with an average age of 8 years, 3 of which were privately owned and 1 that belonged to the National Veterinary, Agrofood, and Food Hygiene School of Loire Atlantique (ONIRIS), Nantes, France. Samples were taken using a closed artificial Missouri vagina (IMV Technologies, L’Aigle, France) filled with water at þ42 C and lubricated with petroleum jelly. The samples were taken using a dummy mount or with a teaser mare next to the dummy. After collection, the sperm was filtered through sterile gauze to eliminate the gel fraction. The volume of the semen was then recorded, the spermatozoal concentration was determined using a photometer (Sperma Cue; Minitube, Germany), and spermatozoal motility was analyzed using computer-assisted semen analysis (CASA). Ejaculates with a concentration of more than 60 106 spermatozoa/ml and a motility of more than 50% [25] were used for this study. 2.2. Extenders 2.2.1. Extraction of LDL The LDLs were extracted using the technique developed by Moussa et al. [21]. Fresh hen eggs were manually broken. Yolks were separated from the albumen and carefully rolled on filter paper to remove chalazas and traces of albumen adhering to the vitelline membrane. The latter was pierced with a scalpel blade, and yolk was collected into a beaker placed on ice. Yolks were diluted with an isotonic saline solution (0.17 M NaCl; w/w) and stirred for 1 hour before being centrifuged at 10,000 g for 45 minutes at 4 C. The supernatant (plasma) was separated from the sediment (granules). Plasma was centrifuged again to remove all traces of contamination with granules; it was then mixed with 40% ammonium sulfate to precipitate out livetins. After 1 hour of stirring at 4 C, the mixture was centrifuged at 10,000 g for 45 minutes. The sediment was discarded and the supernatant dialyzed against distilled water to eliminate the ammonium sulfate. Finally, the solution was centrifuged (10,000 g, 45 minutes, 4 C) and the LDL-rich supernatant was collected. 2.2.2. Preparation of the Extenders We prepared 10 different extenders: INRA 82 with 2% egg yolk (control), INRA 96 (control; IMV Technologies, L’Aigle, France), and INRA 82 with 8 different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), used in centrifugation of the semen. A second series of the same 10 extenders was then prepared but with the addition of 2.5% glycerol (Table 1); these were used in the freezing of the semen.
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Table 1 Composition of different extenders for the centrifugation and freezing of equine sperm, with and without glycerol Composition
LDL 0.25%
General composition
Glucose 2.5 g; lactose 0.15 g; raffinose 0.15 g; sodium citrate 0.025 g; potassium citrate 0.041 g, Hepes 0.476 g; penicillinestreptomycin 0.5 ml; distilled water 50 ml; skimmed milk 50 ml. e e e e e e e e 2 0.680 1.360 2.041 2.721 5.44 8.165 10.88 13.6 e 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 335 334 334 336 335 334 335 338 368
e e 100 ml 301
2.5 100 ml 626
2.5 100 ml 651
Egg yolk (ml) LDL (g) Total volume (QSP) Osmotic pressure without glycerol (mOsm/kg) Glycerol (ml) Total volume (QSP) Osmotic pressure with glycerol (mOsm/kg)
LDL 0.50%
2.5 100 ml 624
LDL 0.75%
2.5 100 ml 627
LDL 1%
2.5 100 ml 626
LDL 2%
2.5 100 ml 626
LDL 3%
2.5 100 ml 627
LDL 4%
2.5 100 ml 628
LDL 5%
2.5 100 ml 628
2% EY Control
2.5 100 ml 764
INRA96 Control e
EY, egg yolk; LDL, low-density lipoprotein; QSP, qunatity sufficient for.
2.3. Freezing The ejaculate was divided into 10 tubes. The sperm in each tube was diluted into equal parts (v/v) at þ37 C with each prepared extender but without glycerol. The tubes were centrifuged at 600 g for 10 minutes to eliminate seminal plasma. The pellet was put back into suspension in the 10 freezing extenders (those containing glycerol) to obtain a final dilution of 100 106 spermatozoa/ml. After dilution, the sperm was cooled from þ37 C to þ4 C in a refrigerated cabinet at þ4 C for 60 minutes. The sperm was then packed into 0.5-ml straws (IMV Technologies), four straws for each extender tested, and sealed with polyvinyl alcohol. The straws were left on a metal ramp for 30 minutes at þ4 C. After equilibration, the straws were frozen by placing them in the vapors 4 cm above liquid nitrogen (120 C) for 10 minutes. Finally, they were immersed in the liquid nitrogen (196 C).
(VSL: mm/s), average path velocity (VAP: mm/s), and amplitude of the lateral head displacement (ALH: mm/s).
2.6. Assessment of Spermatozoal Integrity The last two straws of each extender tested were thawed in a water bath at þ37 C for 30 seconds. Four tests were conducted.
2.5. Motility Analysis
2.6.1. Assessment of Plasma Membrane Integrity: Hypo-Osmotic Test The integrity of the plasma membrane was assessed using the hypo-osmotic (HOS) test to observe the transport of liquids across the plasma membrane [13]. For this test, 25 ml of sperm was diluted in 25 ml of a hypo-osmotic solution (100 mOsm/kg H2O) prepared with 75 mM fructose and 25 mM tri-sodium-citrate in distilled water. After incubation at þ37 C for 60 minutes, a smear was made with 5 ml of the mixture. The slides were then observed under the microscope (CK2, ULWCD 0.30; Olympus) at a magnification of 400 (200 spermatozoa were observed). Spermatozoa were classified as positive or negative depending on the presence or absence, respectively, of a curled flagellum.
Semen was analyzed twice: before freezing (after incubation for 60 minutes at þ4 C in the different extenders) and after thawing (after incubation at 37 C for 10 minutes). An analyzer (Hamilton Thorne) with Ceros 12 software (IMV Technologies, Aigle, France) was used. After thawing, 2 ml of semen were sampled using a micrometric pipette and placed in the chamber of a Leja cell (CryoBio system; IMV Technologies). To prevent thermal shock, the cells were initially placed on a heated stage at þ37 C and observed at a magnification of 200. Five fields were chosen at random over the chamber and analyzed, a mean was then taken. Twenty images per second were taken. The objects present in the cell were recognized as being spermatozoa as a function of their size and brightness. The following parameters were analyzed: motility (% motile spermatozoa), progressively motile spermatozoa (% progressive sperm cells: average path velocity higher than 50 mm$s1, and straightness of track higher than 70%), curvilinear line velocity (VCL: mm/s), straight line velocity
2.6.2. Assessment of Acrosome Integrity: Pisum sativum Agglutinin-Fluorescein Isothiocyanate Test The integrity of the acrosome was assessed using the Pisum sativum agglutinin-fluorescein isothiocyanate (PSAFITC) test, in accordance with the technique described by Mendoza et al. [19]; 5 ml of the sperm being studied was used to make one smear. After being air dried, the slide was immersed in a solution of 100% methanol for 15 minutes to fix and permeabilize the membrane. After drying, the slide was incubated with 200 ml of a solution of PSA-FITC (50 mg/ml) for 10 minutes at room temperature in the dark. The slide was then rinsed and immersed in distilled water for 15 minutes and then air dried at þ4 C. The evaluation was conducted with fluorescence microscopy (DM-IRB model; Leica Microsystems, Wetzlar, Germany). Two hundred spermatozoa were observed. The positive spermatozoa presented with selective staining of the acrosome (intact spermatozoa), while the negative sperm did not take up the stain.
2.4. Thawing Thawing was achieved by immersing two straws of each extender in a water bath at þ37 C for 30 seconds [25].
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2.6.3. Evaluation of DNA Structure: Acridine-Orange Test The structure of the chromatin was assessed using the acridine-orange (AO) stain test. For this test, 150 ml of the sperm was diluted with 4 ml of PBS that had been incubated in a water bath at þ37 C. The tubes were centrifuged at 500 g for 10 minutes. Then, 100 ml of the pellet was mixed with 100 ml of the TNE buffer solution (0.01 M TrisHCl, 0.15 M NaCl, and 1 mM disodium EDTA, pH ¼ 7.4). The suspension of sperm was then mixed with 400 ml of Triton solution (0.1% Triton, 0.15 M NaCl, 0.08 M HCl, pH ¼ 1.2). After 30 seconds of incubation, 1.2 ml of acridine orange (0.2 M Na2HPO4, 1 mM disodium EDTA, 0.15 M NaCl, 0.1 M citric acid, and 6 ml/ml acridine orange, pH ¼ 6.0) were added to the mixture, followed by incubation in the dark for 5 minutes to enable fixation of the AO. Finally, 5 ml of suspension was spread on a slide and covered with a coverslip. The slides were observed with fluorescence microscopy (DM-IRB; Leica) at a wavelength of 520 nm. For each slide, 200 spermatozoa were counted and classified into two groups according to the color of their head: green staining indicated the presence of native (double-stranded) DNA, and orange staining indicated the presence of denatured (single-stranded) DNA. 2.6.4. Evaluation of Spermatozoal Morphology: Spermac Stain Spermatozoal morphology was assessed using the Spermac stain (Minitube). The reagents were placed in 20ml flasks. A smear was made with the semen to be evaluated and left to air dry for 5 minutes. The slide was then fixed by immersion in the flask containing the fixative for 5 minutes. After being air dried for 1 hour, the slide was rinsed five to six times with distilled water, and excess water was removed with absorbent paper. The slide was then plunged into reagent A (red) to be stained for 2 minutes and rinsed with distilled water. A second staining was performed for 1 minute in reagent B (pale green). After rinsing, a third and final staining was performed in a bath containing reagent C (green) for 1 minute before being rinsed again. The slides were observed under the microscope with oil-immersion objective. The acrosome stains dark green, the nucleus red, the equatorial region pale green, and the intermediate section of the tail green.
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Table 2 Motile and progressive spermatozoa in the various extenders Extender
M (%)
0.25% LDL 0.50% LDL 0.75% LDL 1% LDL 2% LDL 3% LDL 4% LDL 5% LDL Egg yolk (control) INRA 96 (control)
76.6 79.7 78.7 78.5 80.4 80.7 78.5 76.8 76.6 72.4
PS (%) 2.1ab 1.9 2.3 2.6 2.1 2.3 2.6b 2.4ab 3.1ab 2.7ab
39.2 41.6 40.8 41.0 43.0 42.3 40.7 39.5 38.8 39.4
1.7ab 1.8 2.1a 2.2a 2.7 2.3 2.1ab 2.0ab 2.4ab 2.1
Table shows mean (SEM) percentages of motile (M) and progressive spermatozoa (PS) in the various extenders at T0 þ 60 minutes, at þ4 C before freezing (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
Two hundred spermatozoa were counted and classified as normal or abnormal. The various types of anomalies were classified according to their location, that is, anomalies of the spermatozoa head, vesicles, damaged acrosome, double heads, and decapitated heads and anomalies of the intermediate piece (swollen) and anomalies of the flagellum (swollen flagellum or cut flagellum). 2.7. Statistical Analysis An analysis of variance (ANOVA) model was used with repeated values according to a linear model with a mixed effect, studied using S-plus statistical software, to determine the influence of the various treatments on motility and progressive spermatozoa, as well as the various motility parameters; these same tests were also applied to the results of the various stains. The equality of variances test using paired results enables a comparison extender by extender. 3. Results 3.1. Motility and Percentage of Progressive Spermatozoa At time 0 (T0), immediately after sperm collection, and before dilution in the various extenders, the mean SD
Fig. 1. Comparison of percentages of motile and progressive spermatozoa. Values are means (SEM). Comparison of the percentages of motile and progressive spermatozoa in various extenders at T0þ10 minutes after thawing (n ¼ 14). (a) P < .05 versus 2% LDL; (b) P < .05 versus 3% LDL.
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motility was approximately 88.7% 6.29% with an average of 42.7% 9.27% progressive spermatozoa for the 14 ejaculates collected. The mean percent of motility before freezing (incubation at þ4 C for 1 hour) was statistically higher (P < .05) with the extenders made with 2% and 3% LDL than with the control extenders made with egg yolk and INRA 96 and with the 0.25% and 5% LDL extenders. There were no significant differences with the 0.50%, 0.75%, and 1% LDL extenders (Table 2). For the progressive spermatozoa, the 2% LDL extender was significantly superior (P < .05) to the control extender made with egg yolk and to the 0.25%, 0.75%, 1%, 4%, and 5% LDL extenders, but there were no significant differences with the INRA 96 extender and the 0.5% and 3% LDL extenders (Table 2). The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant differences were observed among these extenders. The 2% and 3% LDL extenders were significantly superior (P < .05) to those obtained with the control INRA 96 and to the 0.25%, 1%, and 5% LDL (Fig. 1). The post-thaw progressive spermatozoa percentages with the 0.50%, 0.75%, 2%, and 3% LDL extenders and the extender containing egg yolk were significantly superior (P < .05) to those obtained in the 0.25% and 5% LDL and the INRA 96 extenders (Fig. 1). 3.2. Study of Movement Characteristics of Spermatozoa Before freezing (incubation at þ4 C for 1 hour), the extenders containing 0.25% and 0.50% LDL gave superior results for VAP, VSL, and VCL. There was a significant difference (P < .05) between these extenders and all of the other extenders in the study. There were no significant differences among the ALH values in all of the extenders (P > .05) (Table 3). After thawing, the best VAP, VSL, and VCL values were obtained with the extenders containing low concentrations of LDL (0.25%, 0.50%, and 0.75%) and the control extender containing egg yolk. These extenders were significantly superior (P < .05) to the other concentrations of LDL and to the INRA96 extender. Again, there were no significant differences (P > .05) in ALH values among any of the extenders (Table 3). 3.3. Assessment of Spermatozoal Integrity 3.3.1. HOS Test Membrane integrity with the extenders containing 0.50%, 0.75%, 1, 2, 3 and 4% LDL were significantly superior (P < .05) to those obtained with the control extender INRA 96, but no significant difference was observed with the control egg yolk extender. The 2 and 3% LDL extenders were significantly superior (P < .05) to those obtained with the 0.25, 1, and 5% LDL (Table 4). 3.3.2. PSA-FITC Test The percentage of spermatozoa with green stained acrosomes (intact) was approximately 83% for all of the extenders. The high concentrations of LDL (4% and 5% LDL) gave percentages significantly lower than those of the 0.5%, 1%, 3% LDL and INRA96 extenders (P < .05). However, no
Table 3 Automated motility parameters before freezing and after thawing Group
VAP (mm/s)
VSL (mm/s)
Before freezing (T0 þ 60 min at 4 C) 0.25% LDL 90.6 1.2ab 67.0 0.6ab 0.50% LDL 90.5 1.6ab 66.1 1.4ab 0.75% LDL 88.0 1.7b 65.0 1.3b 1% LDL 86.7 2.1 64.1 1.6 2% LDL 86.0 1.7 64.0 1.6 3% LDL 85.8 1.7 63.4 1.4 4% LDL 85.2 1.8 63.2 1.4 5% LDL 84.6 1.9 62.7 1.2 Egg yolk 87.8 1.4a 64.6 1.2 (control) INRA 96 76.6 2.1ab 57.6 1.5ab (control) After thawing (T0 þ 10 min at 37 C) 0.25% LDL 69.8 3.1ab 59.0 2.6ab 0.50% LDL 68.7 2.9ab 58.0 2.4ab 0.75% LDL 69.9 2.3ab 59.8 1.9ab 1% LDL 66.3 3.1 55.8 2.7 2% LDL 65.6 2.9 55.5 2.4 3% LDL 64.6 2.3 55.0 1.7 4% LDL 65.5 1.9 56.3 1.4 5% LDL 61.7 2.4ab 52.6 1.8b Egg yolk 70.8 2.6ab 59.8 2.1ab (control) INRA 96 53.6 2.0ab 45.4 1.9ab (control)
VCL (mm/s)
153.8 154.9 152.0 152.0 149.7 150.0 148.1 146.4 152.0
2.7ab 2.4ab 2.2 2.3 2.1 2.3 2.5 3.5 1.7
141.5 3.3ab
115.8 115.4 115.0 112.1 110.2 109.0 108.6 104.2 117.0
4.3ab 4.4ab 3.2ab 4.5 3.8 4.1 3.4 3.9ab 3.8ab
95.6 3.6ab
ALH (mm/s) 5.7 5.9 5.8 6.0 5.9 5.9 5.8 5.7 5.9
0.12 0.11 0.10 0.11 0.13 0.14 0.14 0.15 0.10
6.0 0.14
4.9 4.8 4.6 4.9 4.7 4.5 4.5 4.6 4.8
0.17 0.17 0.16 0.17 0.18 0.20 0.18 0.14 0.20
4.8 0.13
ALH, amplitude of lateral head displacement; VAP, average path velocity; VCL, curvilinear line velocity; VSL, straight line velocity. Mean (SEM) automated motility parameters before freezing and after thawing (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
significant differences were observed between the different concentrations of LDL and the control egg yolk extender (P > .05) (Table 5). 3.3.3. AO Test According to the equality of variances on paired observations test, there was no significant difference between the different extenders with the AO test. DNA was preserved in 98% of the spermatozoa in all of the extenders. 3.3.4. Spermac Stain After thawing, over 70% of the spermatozoa did not present any anomalies; 78% for the extenders containing 0.25% and 2% LDL and the control extender containing egg yolk (Fig. 2). All of the values were similar, except for the 5% LDL extender, which gave a percentage that was significantly lower (P < .05) than that of the other extenders. The
Table 4 Percentages of intact plasma membranes detected by the HOS test after thawing as a function of the extenders LDL LDL LDL LDL 0.25% 0.50% 0.75 1% SEM 27.0ab 31.3 SEM 1.7 1.9
LDL LDL LDL LDL 2% 3% 4% 5%
EY 2%
INRA 96
29.5 28.9ab 32.3 32.4 29.7 26.5ab 31.3 23.3ab 2.1 1.5 1.2 1.7 2.1 1.8 1.5 1.4
Mean (SEM) percentages of intact plasma membranes detected by the HOS test after thawing, as a function of the extenders (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
D. Moreno et al. / Journal of Equine Veterinary Science 33 (2013) 1068-1075 Table 5 Percentages of spermatozoa with an intact acrosome (PSA-FITC test) after freezing-thawing in the various extenders LDL LDL LDL LDL 0.25% 0.50% 0.75% 1% SEM 84.3 SEM 1.0
85.8a 85.0 1.4 1.1
LDL 2%
LDL 3%
LDL 4%
LDL 5%
EY 2%
INRA 96
84.5 84.0 84.7 83.2b 82.2b 83.5 84.8 0.8 1.2 0.8 0.9 1.3 1.3 1.3
Mean (SEM) percentages of spermatozoa with an intact acrosome according to PSA-FITC test results after freezing-thawing in the various extenders (n ¼ 14). a P < .05 versus 2% LDL. b P < .05 versus 3% LDL.
most common types of anomalies in all the extenders were those of the flagellum, followed by the head and intermediate piece. 4. Discussion The results of this study show that LDL at a concentration of 2% and 3% significantly improve spermatozoal motility and the percentage of progressive spermatozoa following the chilling of equine semen in comparison with the extender containing egg yolk and INRA 96 supplemented with 2.5% glycerol after 1 hour at þ4 C (P < .05). After the freeze-thaw process, the rates were also superior, but not significantly so, than those obtained with the egg yolk extender. With the higher concentrations of LDL (4% and 5%), the plasma membrane and acrosome integrity were less well preserved, and the percentage of spermatozoa with anomalies increased. This confirms previous findings where the best results for the cryopreservation of stallion sperm were achieved with 2% egg yolk, whereas in bulls and dogs, a concentration of 20% was needed. With LDL, the best results for freezing were obtained with a concentration of 8% in the bull [4] and 6% in the dog [6]. In the horse, LDLs have a cryoprotective effect but at a concentration between 2% and 3%. The exact concentration could be determined with a higher number of stallions, possibly 2.5%.
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Khlifaoui [16] has already shown the beneficial effect of LDL for the cryopreservation of equine sperm but at a concentration of 0.5%. For most stallions in this study, the best results were obtained with 2% or 3% LDL; however, in 1 stallion, the 0.5% concentration gave the best results. At the time of sampling, spermatozoal motility was very good, at approximately 90%. The effect of the LDL concentration of the extender could be linked to the composition of the seminal plasma of each stallion, which in turn affects the percentage of motile spermatozoa; this hypothesis needs further investigation. 4.1. Assessment of the Integrity of the Plasma Membrane: HOS Test Spermatozoa with a swollen tail (flagellum) have an intact plasma membrane. Results show that all of the extenders used provided good protection of the flagella plasma membrane, with the exception of INRA 96 supplemented with glycerol. The extenders containing 2% and 3% LDL provided the same level of protection as the egg yolk extender. Because of the low quantity of LDL to be added to the various freezing extenders, the highest percentage of recovered spermatozoa after freezing and thawing was obtained with the 2% LDL extender; while the control extender made with 2% egg yolk has an equivalent LDL concentration of 0.66% LDL. 4.2. Assessment of the Integrity of the Acrosome: PSA-FITC Test It has been proposed that egg yolk contains progesterone [10], which is responsible for the capacitation of spermatozoa and is therefore prejudicial to the preservation of spermatozoa during freezing. This test is justified by the fact that mammalian spermatozoa are incapable of fertilizing the oocyte immediately after ejaculation. It is the fixation of the spermatozoon to the zona pellucida that triggers the acrosome reaction,
Fig. 2. Evaluation of spermatozoal morphology. Values are means (SEM). Comparison of the percentage of spermatozoa without anomalies (Specmac test) in the various extenders after thawing (n ¼ 14). (a) P < .05 versus 2% LDL; (b) P < .05 versus 3% LDL.
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making it capable of fertilization. Spermatozoa that spontaneously undergo an acrosome reaction after ejaculation or following freezing are incapable of fixing to the zona pellucida and are therefore unable to fertilize the oocyte [32]. The extenders containing 2% or 3% LDL provided good protection of acrosome integrity, possibly via a direct action through the exchange or repair of acrosomal membrane phospholipids or possibly simply because the extender has a lower progesterone content than egg yolk because of the filtering effect of the dialysis membrane. The progesterone found in egg yolk plays a role in the capacitation of spermatozoa in cattle [31], horses [2], and humans [20,30]. It appears to act via an extragenomic action on human spermatozoa, via the secondary activation of calcium channels leading to an increase in intracellular Ca2þ, which may be responsible for the capacitation of spermatozoa [2]. Such a mechanism has also been demonstrated in the dog [31]. 4.3. Assessment of DNA Integrity: Acridine Orange Test The integrity of the DNA or chromatin is an important factor when assessing spermatozoa fertility. For this study, AO proved to be effective despite the application of fluorescence microscopy. The results obtained are very good, greater than or equal to 98%. This suggests that LDL provides good protection for cellular DNA during the freeze-thaw process of equine semen. Therefore, extenders containing 2% or 3% LDL are good equine semen preservation agents in comparison with extenders containing egg yolk and INRA 96 supplemented with glycerol. This result was found in all analyses performed and concurs with research undertaken in bulls and dogs [4,6,21]. 4.4. Assessment of Acrosome Integrity: Spermac Test After thawing, 78% of the spermatozoa did not present with any abnormalities with the 0.25% and 2% LDL and egg yolk extenders. The 2% LDL extender seems to provide spermatozoal protection during the freeze-thaw process similar to the EY extender. 5. Conclusions This study demonstrated that extenders containing 2% and 3% LDL give percentages of motile spermatozoa during chilling that are significantly superior to those of the current standard extenders made with egg yolk and INRA 96. After thawing, LDL has a very good cryoprotective effect; at a concentration of 2%, it shows cryoprotective qualities similar to the current extender containing egg yolk and results that are superior to the INRA96 extender and other concentrations of LDL. Acknowledgments The authors thank Myriam Larrat, Sylvie Anezo, and Gérard Chatagnon from the Department of Biotechnology of Reproduction Nantes for technical assistance.
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