Development of extender based on soybean lecithin for its application ...

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Theriogenology 74 (2010) 663– 671 www.theriojournal.com

Development of extender based on soybean lecithin for its application in liquid ram semen P. de Pazc,*, M.C. Estesoa, M. Alvareza, M. Matac, C.A. Chamorrob, L. Anela a

Animal Reproduction and Obstetrics, University of León, 24071, León, Spain b Veterinary Anatomy, University of León, 24071, León, Spain c Cell Biology, University of León, 24071, León, Spain

Received 26 June 2009; received in revised form 16 March 2010; accepted 25 March 2010

Abstract The soybean lecithin is used as a phospholipids source for the commercial extenders available for freezing bull semen which allows replacing the traditional membrane protective of animal origin (egg yolk). These extenders have been tested for freezing semen in various livestock species but specific adjustments cannot be made due to trade protection. The aim of the present study was to develop a soybean-based extender analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. Its effect on the quality of liquid ram semen was also studied. Different TES-Tris-Fructose-based extenders were prepared using two soybean types (S20 and S95) differentiated by their lipid composition (complex or simple, respectively). These extenders were made up in two temperatures: 20 °C (PT20) or 37 °C (PT37); centrifuged and filtered at 20 °C and stored at 15 °C or 5 °C (ST15 and ST05) for several periods (from 6 hours to 7 days). Three different concentrations of soybean (0.5%, 2%, and 3.5%) were evaluated for each extender. The amount and nature of phospholipids present in the extender were evaluated by high performance liquid chromatography (HPLC) method according to the different parameters applied in their preparation. In general, the highest quantity of phospholipids is observed in S20 extender. Centrifugation-filtration process during the extender preparation reduces by 50% the quantity of phospholipids in medium for different experiments. The quantity of phospholipids was not affected significantly by preparation temperature in S20 extender. Storage temperature affects the phospholipids present in the extender (S20 and S95) with minimum values for the storage at 5 °C. As for the storage time, both extenders (S20 and S95) showed a stable quantity of phospholipids in the course of the time, for 2 days at 15 °C and for 7 days at 5 °C. The extender obtained with a higher concentration of soybean (3.5%) showed a higher content of phospholipids under different conditions tested. Finally, sperm motility and viability in new extenders were analyzed. We observed that the sperm quality is not affected by storage temperature for S20 extender. Sperm motility was higher in S20-2% extender and control (UL). Our results suggest that a soybean lecithin extender obtained from S20 soybean at 20 °C, centrifuged and filtered, preserve the sperm motility and viability at 15 °C and 5 °C as an egg-yolk extender. © 2010 Elsevier Inc. All rights reserved. Keywords: Ram sperm; Extender; Soybean lecithin; Phospholipids.

1. Introduction The modern cattle industry worldwide is based on the use of artificial insemination (AI) and frozen semen * Corresponding author. Tel.: ⫹0034987291204; fax: ⫹0034987291322. E-mail address: [email protected] (P de Paz). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.022

that has permitted an accelerated rate of genetic selection and improvement in animal production. AI is less commonly used in ram than in other domestic species because of the difficulty in applying frozen-thawed sperm [1]. The AI commercial programs can be classified in two categories [2]: those using refrigerated semen (15 °C) by intra-cervical deposition (vaginal) and

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those using thawed sperm by intrauterine deposition (laparoscopy). Thus, ovine AI fertility depends on the interaction between the sperm preservation method and the application technique [3]. At present, the most useful method for ovine intracervical AI is the application of refrigerated semen at 15 °C during 6 – 8 h. However, liquid semen must have a minimum shelf-life of between 2 and 4 days in order to be used in distant locations and to cover a large number of ewes in a short period of time [4]. Egg yolk-based extenders are known to be practical and efficient in protecting ram spermatozoa against cold shock during storage before AI [1,5,6]. Nevertheless, the wide variability in their composition and the risk of microbiological contamination and disease transmission are important handicaps to be resolved [7]. Furthermore, the greater viscosity and the presence of particulate debris in extenders due to egg yolk globules interferes in the microscopic assessment of the sample [8] and could be the cause of reduced fertility [9]. Therefore, a well-defined and pathogen-free substitute of non-animal origin for egg yolk should be included in the extender composition. An alternative to egg yolk in extenders for ram semen could be soybean lecithin [2], which is present in several commercial extenders developed for bull semen. Some authors have tested these commercial extenders of bull semen for the cryopreservation of ram semen [7,9,10,11]. The fertility using these extenders have been tested for liquid storage of equine semen [12] and for freezing semen in bull [13,14,15,16] and ram [17,18,19] but it is not possible to make specific settings in your composition due to trade protection. One problem when soy lecithin is added to a base extender is obtaining a homogeneous solution, because soy lecithin is insoluble in water solutions and originates emulsions. This could prove to be a handicap when the extender is used after hours or days of storage at 5 °C and 15 °C, which is required in the field when AI is applied with refrigerated semen. Temperature conditions, preparation methods, and storage time could affect the emulsion formation and phospholipids availability in the extender. The aim of the present study was to develop an extender based on soybean lecithin to replace traditional egg yolk extenders analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. We analyzed the extenders and storage conditions assessing the quantity and nature of phospholipids and the motility and viability in liquid ram semen. These data could provide the

basis for new extenders on those species that lack specific formulas. 2. Materials and methods To obtain a stable soy lecithin-based extender with high phospholipids concentration, we used two soybean types (S95 and S20) comparing different preparation temperatures (37 °C and 20 °C), various storage temperatures, and time (at 15 °C for up to 48 h and at 5 °C for up to 7 d) and testing three concentrations of soybean lecithin (0.5%, 2%, and 3.5%). All the chemicals, unless otherwise specified, were obtained from SigmaAldrich® (Madrid, Spain) in the reagent grade. 2.1. Preparation of the extenders We tested two types of soy: Soy 95% (L-␣-Phosphatidilcholine, soy 95%; S95) and Soy 20% (L-␣Phosphatidilcholine, soy 20%; S20) both from Avanti Polar Lipids® (Alabaster, Alabama, USA). S95 was composed of phosphatidylcholine (PC) and a minority phospholipid, lysophosphatidylcholine (LPC). Soy 20% was composed of PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE), and LPC. A TES-Tris-Fructose buffer solution [TES solution (325 mOsm/kg) titrated to pH 7.2 with Tris solution (325 mOsm/kg), and with 4% final volume of D-fructose solution (325 mOsm/kg)] was used as base extender according to Anel et al [20] and, except for the concentration experiment, 2% solutions of S95 and S20 were prepared. 2.2. Quantitative and qualitative analyses of phospholipids (chromatography) The quantitative and qualitative analyses were carried out by high performance liquid chromatography (HPLC) method. We tested each samples three times in the HPLC equipment (Alliance Waters 2690 Separation Module) which consists of a LC-10A pump, an injector with a 20 ␮L sample loop, and an ultraviolet detector working at a wavelength of 203 nm (Waters 996 Photodiode Array Detector; Waters, Millford, MA). The chromatographic data were acquired by chromatography working station (Millenium32 version 3.05.01, Waters, Millford, MA). The separation was performed on a 150 ⫻ 4.6 mm column packed with 5 ␮m home made silica. The column (Tracer Excell 120) and precolumn were obtained from TEKNOKROMA® (Barcelona, Spain). The samples were diluted in chloroform (1:0.250 v/v), washed four times by centrifugation at 3500 ⫻ g for 10 min, and the pellet was then analyzed. A mixture

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of 1% methanol and 1.6% “85%” phosphoric acid in acetonitrile (Romil®, Waterbeach, Cambridge) was used as mobile phase. The flow-rate of the mobile phase was set at 2 mL/min. All experiments were carried out at a column temperature of 20 °C; and at a sample temperature of 4 °C. The quantitative and qualitative analyses were performed by using external standard based on the height of the peak and known retention time, respectively. For the calibration process we used phospholipids standards of known concentrations. The standards and samples were diluted in chloroform. The quantity of phospholipids (g/L) was calculated as the area under the peak in HPLC graphics. Four replicates were analyzed in each experimental procedure. 2.3. Experimental design 2.3.1. Experiment 1: Effect of the preparation temperature, the storage temperature, and the time on the amount of phospholipids in the emulsion We used two types of soy (S95 and S20) using two preparation temperatures 20 °C (PT20) and 37 °C (PT37) to obtain four extenders. A total volume of 100 mL was prepared for each extender. In Experiment 1A, 2% soybean (S95 or S20) was added to the base extender heated to 37 °C (PT37). The samples were gently shaken in a magnetic stirrer until a homogeneous emulsion was formed and phospholipids content was then analysed at 37 °C (37_PRE-C sample). The samples were left at room temperature until they reached 20 °C when the content of phospholipids was tested again (20_PRE-C sample). After, the extenders were centrifuged at 3500 ⫻ g for 30 min and two parts were observed: the top and the bottom. In Experiment 1B, the top of extenders (S95 or S20) was collected and then filtered twice: first through a 3 ␮m pore (Cellulose Nitrate Filter, 11302-25-N, Sartorius, Goettingen, Germany) and then by 0.45 ␮m pore (Cellulose Acetate Filter, 11106-25-N, Sartorius, Goettingen, Germany). At this point, we tested again the phospholipids in the extenders (20_POST-C sample) and two tubes per sample (containing 9 mL) were stored in vertical position. In the following experiments, the extenders were always centrifuged and filtered (POST-C samples). After six hours of storage, we observed again two fractions in S95 extender: the light (LF) and the milky (MF) fractions (Fig. 1). In Experiment 1C, we extracted 1 mL of each fraction to test the phospholipids. In Experiment 1D, the second tube of S95 extender was gently shaken in order to obtain a homogenous extender (H), and 1 mL of this extender was also extracted to test the phospholipids content. The S20 extender shows a

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Fig. 1. A: Two fractions were observed in S95 extender after resting 6 hours in storage: the light fraction (LF) and the milky fraction (MF). B: The same sample after a gently shaken in a magnetic stirrer.

homogeneous appearance (H) and 1 mL of sample was extracted to this experiment. In this trial, the control sample of each soybean is the phospholipids content in extender immediately after centrifugation-filtration at 20 °C (CONTROL). The other samples were stored at two different temperatures: 15 °C (ST15) for 48 h and 5 °C (ST5) for 7 d, the latter two according to the ideal handling schedule of ram semen refrigeration. These extenders were rated to quantify the phospholipids according to the following schedule (Experiment 1E): 6 (D0) and 48 h (D2) at 15 °C, and 6 (D0), 48 (D2), 96 (D4) and 138 h (D7) at 5 °C. A specific assay at ⫺80 °C was carried out with S20 extender stored for 24 d to explore the stability of frozen soybean extender. 2.3.2. Experiment 2: Concentrations assay Three different concentrations of soybean (S95 and S20) were assessed: 0.5, 2, and 3.5% at room temperature (20 °C). We tested phospholipids after filtration in the homogeneous fraction at 48 h (15 °C and 5 °C). 2.3.3. Experiment 3: Sperm motility and viability assays The protective effect of three different extenders defined in previous trials was evaluated by a sperm viability and motility assay using an UL egg yolk-based extender [13] as control. Spermatozoa were incubated in each of the four extenders to a final concentration of 100 ⫻ 106 cell/mL, at 15 °C or 5 °C for 6 h (D0) and then sperm quality was evaluated. 2.3.3.1. Semen collection and initial evaluation. Three adult Churra rams of proven fertility were used. Semen was collected by artificial vagina (40 – 42 °C) during

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the breeding season. Three biweekly sessions of semen collection were done and semen from each session was pooled. Each ejaculate had previously been kept in the collector tube at 35 °C during initial evaluation of semen quality. The volume (using a collector tube graduated in millilitres), mass motility (warming stage at 37 °C, 40x; score: 0 –5) and sperm concentration (by the photocolorimetric method, 540 nm, on a specific calibrated scale) were determined. Only ejaculates of good quality were pooled (volume: ⱖ 0.5 mL.; mass motility: ⱖ 4; sperm concentration: ⱖ 3000x106 spz/mL). 2.3.3.2. Motility. Sperm motility was determined with a computer-assisted motility analyzer (CASA system) using an optical phase contrast microscope [Nikon Labophot-2 equipped with negative phase contrast objectives (10x) and a warming stage at 37 °C], a Sony XC-75CE camera and a PC with the Sperm Class Analyzer software (SCA2002; Microptic, Barcelona, Spain). Samples were diluted (10 –20 ⫻ 106 cells/mL) in HEPES-buffered saline solution (HEPES 20 mm/L, 197 mmol/L NaCl, 2.5 mmol/L KOH, 10 mmol/L glucose; pH 7; 320 mOsm/kg), and warmed on a 37 °C plate for 5 min. Then, a pre-warmed Makler counting chamber (10 ␮m depth) was loaded with 5 ␮L of sample. The proportions of total motile (TM, %), progressive motile (PM, %) were recorded. At least five sequences were saved and analysed afterwards. 2.3.3.3. Viability. Sperm viability was assessed using the LIVE/DEAD® Sperm Viability Kit (Invitrogen, Barcelona, Spain) according to the manufacturer’s instructions. Semen sample was diluted in HEPES-buffered saline solution containing bovine serum albumin. A 50-fold dilution of the SYBR 14 stock solution (Component A) in buffer was prepared and 5 ␮L of diluted SYBR 14 dye (from step 2) was added to 1 mL sample of diluted semen and incubated for 5–10 min at 36 °C. 5 ␮L of Propidium Iodide (Component B) was added to the 1 mL sample of diluted semen and incubated for an additional 5–10 min. The samples were analyzed by flow cytometry (FACScalibur cytometer, Becton Dickinson Immunochemistry Systems; San Jose, CA, USA). 10,000 events with a flow rate of ⬃200 cells/s were analyzed per sample. For data analysis, we used the percentage of viable spermatozoa (SYBR 14 ⫹ / Propidium Iodide -). 2.4. Statistical analysis Data were analyzed using the SAS™ package V.9.0. In Experiments 1 and 2, the amount of phospholipids was the main variable and a GLM procedure was used

to evaluate the effect of various parameters: soy lecithin source (S20, S95), preparation temperature (37 °C, 20 °C), filtration process, fractions (LF and MF in S95), storage temperature (15 °C, 5 °C), storage time (4, 48, 96, 142 h) and soybean concentration (0.5%, 2%, 3.5%). In Experiment 3, the effect of various extenders on motility and viability of spermatozoa was analyzed using a GLM procedure. Values were considered to be statistically significant at p ⬍ 0.05. 3. Results In Experiment 1, we evaluated the preparation of extender from two types of soybean lecithin (S20 and S95) at two preparation temperatures (PT20 and PT37) and one storage temperature (ST20). The amount of phospholipids (g/L) in the various extenders before (PRE-C) and after (POST-C) centrifugation-filtration was quantified (Table 1) and we observed a loss of approximately 50% of the phospholipids in the extenders after this preparation stage. There were quantitative differences between the three types of phospholipids present in the S20 extender in each of the two preparation temperatures (Fig. 2). This extender showed a higher proportion of PC than PE and PI, respectively, at PT20 and PT37 temperatures. The quantity of PC was greater in the S20 extender than in the S95 extender at PT20 (6.0 vs. 4.9) and at PT37 (5.7 vs. 3.7). The presence of phospholipids (g/L) in the dilution extender at different storage temperatures depended on the type of soybean and preparation temperatures (Fig. 3). We observed significant differences between the two types of soybean analyzed, obtaining the highest values of phospholipids in the S20 extender in comparison with S95 for both preparation temperatures. A similar amount of phospholipids in the S20 extender was observed in both preparation temperatures PT20 and PT37 (range 9.6 – 8.9). However, the S95 extender prepared at 20 °C showed a greater amount of phospholipids than that prepared at 37 °C (4.8 –2.6 vs. 2.7–1.8 at PT20 and PT37, respectively). Experiment 2 evaluated the effect of storage time at two temperatures (15 °C and 5 °C) on the concentration of phospholipids (g/L). The amount of phospholipids in both extenders (S20 or S95) did not change during storage for 2, 4, or 7 days at 5 °C (Fig. 4) or by storage for 2 or 4 days at 15 °C (Fig. 5). However, in all cases, the amount of phospholipids was always higher in the S20 extender than in S95.

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Table 1 Effect of centrifugation-filtration (PRE-C vs. POST-C) on the amount of phospholipids (mean of four replicates ⫾ standard error in g/L) in TES-Tris extender prepared with soy lecithin depending on the type of soybean (S20-S95) and the preparation temperature (PT20-PT37). Preparation temperature

Sampling stage

S20 Mean

PT20 PT37

a,b A,B

␣,␤

20_PRE-C 20_POST-C 37_PRE-C 20_PRE-C 20_POST-C

22.5 11.2 28.1 24.0 11.4

S95 ⫾ SE ⫾2.0 ⫾0.2 ⫾5.6 ⫾2.6 ⫾0.3

aA␣ bA␣ a␣ aA␣ bA␣

Mean

⫾SE

7.4 4.8 5.8 4.7 2.7

⫾0.2 ⫾0.5 ⫾1.5 ⫾1.0 ⫾0.2

aA␤ bA␤ a␤ aB␤ bB␤

In the same column, different superscripts indicate that PRE-C and POST-C means differ within each preparation temperature PT (P ⬍ 0.05). In the same column, different superscripts indicate that means differ between temperatures of preparation (P ⬍ 0.05). In the same row, different superscripts indicate that columns differ (P ⬍ 0.05).

When the S95 extender was stored for several hours, the milky fraction had a higher concentration of phospholipids (range 5.0 –2.5) than light fraction (range 2.0 –1.2) for both storage temperatures (Table 2). The temperature of preparation did not significantly influence the presence of phospholipids in the two fractions. The homogenized suspension showed no significant differences in the two temperatures tested (range 3.4 – 2.5). To define a suitable extender, we tested three concentrations each of soybeans (0.5, 2, and 3.5%) that were prepared at 20 °C, filtrated, and stored at 15 °C or 5 °C for 48 h (Table 3) and were then evaluated to calculate the phospholipids amount (g/L). In all cases significant dif-

Fig. 2. Quantity of various phospholipids (mean of four replicates ⫾ standard error in g/L) in an extender, immediately after centrifugation-filtration at 20 °C (POST-C), depending on the type of soybean (S20-S95) and the preparation temperature: 20 °C (PT20) or 37 °C (PT37). (PC: PhosphatidylCholine, PI: PhosphatidylInositol, PE: PhosphatidylEthanolamine).

ferences between the S20 and S95 extenders were observed for different temperatures and concentrations. A particular assay at ⫺80 °C was carried out with the S20 extender, stored for 24 d, and then the amount of phospholipids (g/L) was analysed (S20 –3.5%: 13.6; S20 –2%: 8.3; S20 – 0.5%: 1.2). In this case, a signifi-

Fig. 3. Evolution of the quantity of phospholipids (mean of four replicates ⫾ standard error in g/L) in each extender after centrifugation-filtration according to storage temperature (15 °C: ST15 or 5 °C: ST05) depending on the type of soybean (S20-S95) and the temperature of preparation (20 °C: PT20 or 37 °C: PT37). A control sample of each soybean obtained at 20 °C immediately after centrifugation is used. a,b Different letters indicate that means of each storage temperature differ between extender for each preparation temperature (P ⬍ 0.05).

a,b Different letters indicate that means for S20 differ within each preparation temperature (P ⬍ 0.05).

A,B

A,B

␣,␤

Different letters indicate that means of PC content for each preparation temperature differ between extenders (P ⬍ 0.05).

Different letters indicate that means of each extender differ between temperatures of preparation (P ⬍ 0.05).

Different letters indicate that means of each extender differ between storage temperature (P ⬍ 0.05).

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Fig. 4. Evolution of the quantity of phospholipids (mean of four replicates ⫾ standard error in g/L) into extender according to the days of storage (Non storage: D0; 2 days: D2; 4 days: D4 or 7 days: D7) at 5 °C depending on the type of soybean (S20-S95) and the temperature of preparation (20 °C: PT20 or 37 °C: PT37). a,b

Different letters indicate that means of each storage day differ between extender for each preparation temperature (P ⬍ 0.05).

lecithin and we evaluated the best conditions for use, handling, and storage of soybean lecithin. Moreover, the protective effects of the new extender on the motility and viability of ram semen were studied and compared with those of egg yolk extender (UL) tested previously by us [13]. The qualitative and quantitative analysis of S20 extender using HPLC shows that the major phospholipids were PC, PE, and PI, and that PC was only significantly concentrated in S95. We observed that the S20 extender contains more total phospholipids than S95 and we think that this difference may be due to the higher viscosity of S95 and to the different phase transition temperatures of the phospholipids. In addition, the particle size of PC is higher than the rest of the phospholipids that form part of the soy lecithin [21] and this lipid species is a majority in S95 soybean. Therefore, the time required for the formation of the emulsion with S95 soybean was shorter in the case of samples prepared at 37 °C compared to samples prepared at room temperature. Previous studies on the handling conditions of soybean lecithin showed that the temperature

A,B

Different letters indicate that means of each extender differ between temperatures of preparation (P ⬍ 0.05).

␣,␤

Different letters indicate that means of each extender differ between storage days (P ⬍ 0.05).

cant reduction of phospholipids content (g/L) was observed in comparison with data obtained for storage at 5 °C and 15 °C. Finally, the effect of different extenders on seminal quality (motility and viability) was studied (Table 4). The S20 –2%, S20 –3.5% and UL extenders showed the best values of sperm motility at 15 °C and 5 °C (TM: interval 77.7–91.0%; PM: interval 42.1–58.9%). Sperm viability did not show significant differences between the various extenders analyzed. In the S20 extenders the storage temperature did not affect sperm motility and viability, however the S95 extender showed the best motility values at 15 °C (TM: 66.5– 47.9% vs. 23.3–9.4% at 5 °C). 4. Discussion The use of chemically defined and animal proteinfree extenders for cryopreservation of semen would circumvent problems associated with traditional egg yolk based extenders, including contamination and variability of composition. In the present study, we proposed the development of a new extender for chilled ram semen based on soy

Fig. 5. Evolution of the quantity of phospholipids (mean of four replicates ⫾ standard error in g/L) into extender according to the days of storage (Non storage: D0 or 2 days: D2) at 15 °C depending on the type of soybean (S20-S95) and the temperature of preparation (20 °C: PT20 or 37 °C: PT37). a,b Different letters indicate that means of each storage day differ between extender at each preparation temperature (P ⬍ 0.05). A,B

Different letters indicate that means of each extender differ between temperatures of preparation (P ⬍ 0.05).

␣,␤

Different letters indicate that means of each extender differ between storage days (P ⬍ 0.05).

P. de Paz et al. / Theriogenology 74 (2010) 663– 671 Table 2 Quantity of phospholipids (mean of four replicates ⫾ standard error in g/L) present in the different phases of the S95 extender (H: Homogeneous suspension, LF: Light fraction, MF: Milky fraction) depending on the temperature of preparation (PT20-PT37) and storage temperature (ST05, ST15). Storage temperature

Phase of extender

PT20

PT37

Mean ⫾ SE

Mean ⫾ SE

ST05

H MF LF H MF LF

⫾ 1.6 ⫾ 0.7 ⫾ 0.2 ⫾ 0.3 ⫾ 0.3 ⫾ 0.2

ST15

aA

2.5 2.5 1.6 3.0 3.9 1.2

aA bA aA aB bA

aA aA aA aA aB bA

a,b

In the same column, different superscripts indicate that means differ within each storage temperature (P ⬍ 0.05). A,B In the same column, different superscripts indicate that means of each fraction differ between storage temperature (P ⬍ 0.05).

and the amount of some types of phospholipids could change the physical characteristics of soybean lecithin [22]. These authors showed that soybean solubility is greater when the handling temperature of the sample is increased. Moreover our results demonstrated that the centrifugation-filtration process significantly reduced the amount of phospholipids present in the S20 or S95 extender for the two preparation temperatures. In order to define an optimal procedure for preparing the soy lecithin extenders, we studied the effect of preparation and storage temperature on the quantity of phospholipids present in the final extender. In S95, the amount of phospholipids was two times greater in the extenders made at 20 °C than in those made at 37 °C.

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These extenders showed a decrease in phospholipids after reaching room temperature. This reduction may be due to two reasons: first, that while they rested at 20 °C, the samples are in contact with atmospheric oxygen, favouring the reaction of lipid peroxidation [23]; and second, that S95 soybean is more soluble at 37 °C and by decreasing the temperature to 20 °C, the viscosity of the sample increases again [22]. A progressive decrease in the quantity of phospholipids is observed when extenders reach storage temperatures of 15 °C and 5 °C after a few hours, being more marked at 5 °C. This fact can be explained by increased viscosity of the extender induced by the lower temperature [22]. In general, both types of soy lecithin extenders remained stable over 2 days of storage at 15 ° C, which was also observed at 5 °C for 7 days. Thus, the extenders at 37 °C have a higher solubility due to temperature, which gradually decreases to 5 °C, when solubility of phospholipids is reduced. In this regard, the content of phospholipids in S20 extender storage for 24 days at ⫺80 ° C was reduced significantly and hence this storage model would not be useful. On the other hand, Wilson and Rinne [24] found that levels of PC, PE, and phospholipids nitrogen decreased and that levels of phosphatidic acid increased, with time prolonged cold storage of soybean cotyledons, due to the higher concentration of phospholipase D, as a result of the dispersion of the enzyme during cell disruption. Similar adverse effects of freezing on specific phospholipids should be considered when using the extenders prepared with soybean for the storage of spermatozoa.

Table 3 Quantity of phospholipids (mean of four replicates ⫾ standard error in g/L) present in an extender according to the type of soybean (S20S95), the concentration of the suspension (0.5%, 2%, 3.5%), and storage temperature. Storage Temperature (Time*) CONTROL (D0)

ST15 (D2)

ST5 (D2)

Concentration (%) 0.5 2 3.5 0.5 2 3.5 0.5 2 3.5

S20

S95

Mean

⫾ SE

5.2 10.7 17.6 3.5 10.6 17.0 3.6 9.3 16.3

⫾ 0.7 ⫾ 1.6 ⫾ 2.3 ⫾ 2.0 ⫾ 0.9 ⫾ 0.2 ⫾ 0.5 ⫾ 0.3 ⫾ 0.7

a

A

␣

b

A

␣

c

A

␣

a

A

␣

b

A

␣

c

A

␣

a

A

␣

b

A

␣

c

A

␣

Mean

⫾ SE

0.6 3.2 5.7 1.0 4.4 6.0 1.0 3.9 6.5

⫾ 0.3 ⫾ 0.4 ⫾ 0.2 ⫾ 0.0 ⫾ 0.0 ⫾ 0.4 ⫾ 0.0 ⫾ 0.4 ⫾ 0.3

a

A

␤

b

A

␤

c

A

␤

a

A

␤

b

A

␤

c

A

␤

a

A

␤

b

A

␤

c

A

␤

A control sample of each soybean obtained at 20 °C immediately after centrifugation is used. * Days of storage (Non storage: D0; 2 days: D2). a,b In the same column, different superscripts indicate that means differ between concentration within each storage temperature (P ⬍ 0.05). A,B In the same column, different superscripts indicate that mean of each concentration differ between storage temperature (P ⬍ 0.05). ␣,␤ In the same row, different superscripts indicate that columns differ (P ⬍ 0.05).

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Table 4 Effect of different extender on motility (TM and PM) and viability of spermatozoa (mean ⫾ standard error in %) in terms of soybean concentration in extender and temperature of storage (D0). (S20: soybean S20 extender at three concentrations 0.5, 2 and 3.5 %; S95: soybean S95 extender at three concentrations 0.5, 2 and 3.5 %; UL: control extender according to Anel et al [13]). Extender Total Motility (%) ST15 S20-0.5 S20-2 S20-3.5 S95-0.5 S95-2 S95-3.5 UL

49.8 86.1 77.7 65.1 66.5 47.9 91.0

Progressive Motility (%) ␣

ST5 ⫾ 12.0 ⫾ 2.6 ⫾ 2.8 ⫾ 0.8 ⫾ 5.3 ⫾ 1.0 ⫾ 1.3

b a c d d b a

33.1 78.4 73.9 23.3 23.3 9.4 85.8

⫾ 7.2 ⫾ 6.8 ⫾ 2.1 ⫾ 6.2 ⫾ 7.8 ⫾ 0.7 ⫾ 3.9

B

*

C

nd

C

nd

B B D A

*** *** *** nd

ST15 9.3 58.9 42.1 22.6 22.2 11.6 52.7

IP-SYBR 14 (Green) (%) ␣

ST5 ⫾ 0.5 ⫾ 5.8 ⫾ 2.1 ⫾ 0.3 ⫾ 5.7 ⫾ 2.1 ⫾ 8.0

b a c d d b a

3.9 48.3 43.8 6.4 8.2 6.8 46.1

⫾ 1.3 ⫾ 7.2 ⫾ 1.5 ⫾ 1.5 ⫾ 1.2 ⫾ 0.3 ⫾ 4.5

B

*

C

nd

C

nd

B B B A

*** ** * *

ST15 66.0 68.4 66.4 63.0 66.8 66.9 70.6

␣

ST5 ⫾ 15.1 ⫾ 13.5 ⫾ 14.3 ⫾ 16.3 ⫾ 13.8 ⫾ 13.6 ⫾ 12.1

a a a a a a a

70.9 71.4 71.1 52.0 70.7 68.1 69.2

⫾ 4.6 ⫾ 10.3 ⫾ 9.8 ⫾ 1.9 ⫾ 7.1 ⫾ 5.4 ⫾ 8.5

A

nd

A

nd

A

nd

B

*

A

nd

A

nd

A

nd

In the same column, different superscripts indicate that means differ between concentration within each storage temperature (P ⬍ 0.05). In the same column, different superscripts indicate that means differ between storage temperature (P ⬍ 0.05). ␣ In the same row, differences between columns within each parameter (nd: non differences, * P ⬍ 0.05 ** P ⬍ 0.01 *** P ⬍ 0.001).

a,b

A,B

During storage at different temperatures, the presence of milky sediment in S95 may be due to the formation of micelles by the phospholipids in the aqueous extender, because when phospholipids are added to water, only a small fraction is dissolved. In general, the higher quantity of phospholipids was observed in MF fraction, where the entire PC of the sample is present. This type of phospholipids precipitates due to its large particle size [22]. We also analyzed the amount of phospholipids present in another sample which was homogenized (H) and we recovered a similar amount of phospholipids, because S95 soybean contains minimum concentrations of PE and PI [21]. In S20 soybean extender, we observed only an homogeneous phase (H) with a higher concentration of phospholipids. The particle size of PC is higher than the rest of the phospholipids that form part of the soy lecithin [21] and so we can explain these different phospholipids concentrations between the S20 and S95 extenders. In order to define the final extenders, we tested three different concentrations of soy lecithin (0.5%, 2%,and 3.5%) with both types of soybeans. Obviously, the amount of phospholipids in the extender increases as the soybean concentration rises in any of the conditions implemented, but it is necessary to evaluate their impact on spermatozoa physiology. Extender S20-2% showed the best values of sperm motility and viability at both 5 °C and 15 °C, which are similar to those of control egg yolk extender (UL). In previous studies, van Wagtendonk-de Leeuw et al. [9] observe that the post thaw motility was significantly lower for bovine semen diluted using a commercial extender based on soybean (Biociphos Plus) compared with extenders based on egg yolk, which is explained by the higher viscosity and the presence of particulate

debris in the Biociphos Plus extender. In this regard, we observed that S95 extender showed lower sperm motility when compared with S20. This fact may also be due to the high viscosity of S95 extender and the higher phospholipids diversity in S20. In general, there were no significant differences in sperm viability in any of the tested extenders at different storage temperatures. 5. Conclusions We propose a TES-Tris-Fructose-soybean lecithin 20% extenders containing 2% or 3.5% of soybean and prepared at 20 °C to store the ram sperm at 15 °C and 5 °C. In subsequent studies, the effect of these extenders on the spermatozoa will be extensively analyzed, and the fertility of liquid ram semen prepared with these soybean extenders will be evaluated. These results may be useful for management of soybean lecithin in seminal extender of species that have no specific commercial products [25] and for this reason should develop new extenders.

Acknowledgements This work was supported by CICYT (AGL200507601/GAN), Junta de Castilla y León, Diputación de León, and ANCHE. Milagros C. Esteso was sponsored by the Juan de la Cierva program from the Spanish Ministry of Science and Innovation.

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