Serious chemical analysis of the seminal plasma was taken up during the present century, leading to the discovery and identification of several substances as the proteins [1]. There is increasing evidence for the multifunctional nature of proteins. It is possible that they may have different functions in the reproductive process [2,3], as biochemical markers for identification of biological properties of animal semen [4,5]. The number of distinct types of proteins normally present in the seminal plasma also varies, but most of the seminal proteins is distributed among four to ten fractions, the proportions of which vary individually and in relation to the time interval intervening between ejaculation analysis [1]. In this respect the high performance the technique of gel filtration chromatography methods is a distinct advantage as study the constituent proteins seminal plasma separating by the molecular size [6-9]. The aim was the chromatography profile to simple study of proteins seminal plasma domestic animals (Stallions, Canine, and Goat), by the technique of gel filtration chromatography, using a Superose 12 HR 10/30, in a fast performance liquid chromatography (FPLC-system).

Animals

Ejaculates of six stallions (5-20 years old) were collected using an artificial vagina (model “Hannover”), the ejaculates of six canines (2-4 year old) were collected by masturbation with a glove or bare hand, and the ejaculates of two goats (2- years old) were by electroejaculator. Semen samples were evaluated for progressive motility with a bright field microscopy (100×). Ejaculates containing a minimum of 50% of spermatozoa with progressive motility were used in the study. All samples were cooled to +5°C with a cooling rate of 1°C/min; such a rate does not induce a cold-shock effect [9-11].

Seminal plasma proteins purifications

To obtain isolated seminal plasma proteins, semen was centrifuged for 30 min at 4°C (600 g). Then, the supernatant was brought to 36% (wt/vol) saturation with ammonium sulfate adjusted to pH 2.0 with 6 mol L-1 HCl, stirred for 30 min, and allowed to stand at 0°C for 30 min [10]. The samples were centrifuged again (600×g, 4°C, 30 min), the supernatant and the ammonium sulfate pellet were dialyzed for 24 h up to a 8.000 dilution factor with 0.5% (by volume) aqueous acetic acid solution using exclusion membrane of 1,000 Da cut-off, and finally the sample were lyophilized, freeze-dried and stored -20°C, according to Vasconcelos et al. [11].

Protein concentration assay was proceeded in test tube, mixing bradford reagent with protein sample (1: 0.1 mL portions, respectively) and strained with coomassie brilliant blue BG-250 [12]. Then, absorbance was measured at 595 nm by spectrophotometry (Shimadzu- 160A).

Chromatography analysis

The seminal samples purifications (0.5 mL; 0.5 mL and 0.2 mL) of stallion, canine and goat, respectively were applied to a Superose 12 HR 10/30 column, molecular exclusion chromatography, equilibrated with 25 mM Tris-HCl (Sigma) with 0.15 M NaCl (Sigma), pH 7.4 at room temperature in a fast performance liquid chromatography (FPLC-system), using a flow rate of 0.5 mL.min^-1 and collected fraction volume of 1.5 mL. The mobile phase was the same solution used for equilibration. The Column was calibrated according to the methods of Andrews [13]. Molecular weight markers (β-amilase 200 kDa, Alcohol desidrogenase 150 kDa, Bovine Albumin 66 kDa, Anidrase carbonic 29 kDa, Cytochrome C 12,4 kDa, Aprotinin 6,5 kDa). Molecular weight markers were like a chromatography marker. Total protein was estimated in each seminal plasma sample by spectrophotometry (Shimadzu-160A); absorbencies were read at 215 nm [14].

Statistical analysis

The linear regression between seminal plasma protein concentration, relative mass and retention time were calculated using the software (Origin version 5®).

Chromatographic calibration

Calibration results of the gel filtration column: y = ax +b; parameters (value, error): a= 5.43, 0.115; b= - 2.175, 0.256; R2= 0.973; (p<0.001), and exclusion volume (Vo) 7.64 min. Our results were according to Andrews methods [15] the experiment error was smaller than 5% for more in the molecular mass the protein.

Chromatography proteins

The proteins chromatography of the stallion seminal plasma were represent by 12 fractions with four different retention time peaks (t_M), according to the chromatogram (Figure 1) in six animals studied. Only four picks graphic areas were selected by showing the relative mass (M_R) increase.

Figure 1: Stallion seminal plasma proteins by chromatography analysis. Superose 12 Column HR 10/30 (FPLC system; 0.025M Tris-HCl buffer pH 7.4; flow rate 0.5 ml/min, sensibilitiy 0.5).

The results emphasized the molecular exclusion technique, characterized by the proteins separation of high molecular weight, proceeding to low molecular weight. The proteins relative mass (M_R), the retention picks value among proteins samples of the seminal plasma were presented (Table 1). This stallion seminal plasma proteins were statistically similar in all animals (p<0.05), as related by Vasconcelos et al. [11].

Table 1: Values of retention time (t_M) and relative mass (M_R) in the stallion seminal plasma proteins after chromatography of molecular exclusion.

The chromatogram of canine seminal plasma proteins (Figure 2) described several retention peaks. The relative mass were calculated according to retention time (23 min; 30.6 min; 37 min), in view of the greater proteins concentration in this range, by spectrophotometer analysis.

Figure 2: Canine seminal plasma proteins by Chromatography analysis. Superose 12 Column HR 10/30 (FPLC system; 0.025M Tris-HCl buffer pH7.6; flow rate 0.5 ml/min, sensibilitiy 0.5).

The retention time and molecular weight averages calculation, of each sample, related to each canine ejaculate suggests the repeatability of the technique represented by the equivalence between the results measurement (Table 2). Where molecular weights can be uncertain, the most likely values indicated often correspond to values suggested by the gel-filtration results. The technique repeatability represented among the measurements results, because there were no statistical difference between animals (p<0.05). This repeatability theory described by Vial and Jardy [15] defined that the equipment calibration were characterized by the use of standard proteins, according to this work, and the effective selective separation protocol of seminal plasma proteins.

Table 2: Values of the estimate of total protein and of retention times (t_M) of the samples of canine seminal plasma.

Chromatographic samples analyze of goat seminal plasma were presented in five peaks (Figure 3). The results showed that proteins variability were seen between the two goats evaluated and the five picks were maintained. This variability can occur since the proteins may have different structural characteristics as a free carbon [15]. However, this technique described a good correlation between relative mass (M_R) and gel-filtration behavior.

Figure 3: Goat seminal plasma proteins by chromatography analysis. Superose 12 Column HR 10/30 (FPLC system; 0.025M Tris-HCl buffer pH7.6; flow rate 0.5 ml/min, sensibilitiy 0.5).

The similarity among the chromatographic profiles obtained from different animals was evaluated through the comparison of the retention time of the signs chromatographically observed and quantified in the Table 3. The related in work with gel exclusion chromatography on G-200 Shephadex performed by Mann [1] showed that two of the basic proteins were purified to apparent homogeneity.

Table 3: Results of time retention (t_M) and values of the estimate of relative mass (M_R) of goat seminal plasma samples.

During the analysis of a new sample, it is possible that not all frequently detected components will be. The failure of their detection indicates that there is a variation in the identifications process concerning mainly technical aspects, like spot excision, digestion, peptide recovery, mass spectra acquisition, and identity assignment [16]. This technique of gel filtration chromatography can be a tool for studying the initial fraction seminal plasma proteins.

A proteomic analysis involves proteins separation and proteins identification as well as characterization of the post-translational modifications. The limitations in a proteomic analysis are of two kinds: (i) those related to the composition of the proteome to be analyzed, mainly concerning protein expression levels and (ii) limitations of the analytical methods [14]. The chromatographic methods can reduce the complexity of the proteins mixtures because of different binding’s principles, and every approach adds a unique resolving power. The standardization using known proteins enable one reason for study area in chromatogram and define a relative protein samples mass present in biological fluids. This technique allows that these molecules interaction with the column matrix do not occur [17,18], concluded that the physico-chemical properties of the technique does not interfere with theprotein structure sample, allowing to monitor the proteins variability presented in the plasma seminal of individuals and species studied.

The authors wish to acknowledge the financial support given to us by the FAPEMIG and the Department of Biochemistry and Immunology of UFMG. In memoriam Marcelo Matos Santoro².