Sperm evaluation and biochemical characterization of cat seminal plasma collected by electroejaculation and urethral catheterization

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Theriogenology 74 (2010) 1396 –1402 www.theriojournal.com

Sperm evaluation and biochemical characterization of cat seminal plasma collected by electroejaculation and urethral catheterization Daniele Zambellia,*, Ramona Raccagnib, Marco Cuntoa, Giulia Andreania, Gloria Isania a

Clinical Veterinary Department, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy b DIMORFIPA—University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy Received 9 July 2009; received in revised form 9 June 2010; accepted 10 June 2010

Abstract This paper aimed to evaluate cat seminal plasma protein profile (with SDS-page) and determine differences in seminal plasma composition from ejaculates obtained using urethral catheterization after pharmacological induction (UrCaPI) and electroejaculation (EE). In addition, this study evaluates whether the recovery method affected seminal plasma protein and zinc concentrations. A single ejaculation was collected from 17 mixed-breed cats by EE (5/21) or UrCaPI (12/21), while 4/21 cats underwent four sperm collections once every four days using EE and UrCaPI techniques alternately. The semen parameters evaluated were: volume, percentage of motility and progressive motility, morphology, and sperm concentration. After centrifugation, the seminal plasma obtained was stored at ⫺80 °C and later used to measure protein and zinc concentrations, and to determine protein profile by SDS-polyacrylamide gel electrophoresis (PAGE). The results obtained indicate that cat seminal plasma protein profile is characterized by many protein bands (⬎30) with a molecular weight ranging from 3.5 to 200 kDa, and that the recovery method influences the seminal plasma protein profile: EE is related to the absence of two proteins (P55 and P14), and alters three protein bands (P200, P80, P28). The collection technique also affected zinc concentration (mg/dL) and protein concentration (g/dL) which were significantly higher (P ⬍ 0.01) in samples collected by UrCaPI; on the contrary the total Zn and protein amount/ejaculate were not significantly different in samples collected by both technique (P ⬍ 0.05). © 2010 Elsevier Inc. All rights reserved. Keywords: Domestic cat; Seminal plasma; Protein; Electrophoresis; Zinc

1. Introduction Seminal plasma is the secretion of the sexual accessory glands released in the urethra at the time of ejaculation to support spermatozoa. It contains proteins, including many enzymes (acid phosphatase, alanine transaminase, alkaline phosphatase, aspartate transaminase), lipids, macroelements [sodium (Na⫹), potassium (K⫹), calcium (Ca2⫹), magnesium (Mg2⫹),

* Corresponding author. Tel.: ⫹39 051 2097572; fax: ⫹39 051 2097568. E-mail address: [email protected] (D. Zambelli). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.06.011

phosphate (P) and chloride (Cl)] and microelements [copper (Cu), iron (Fe) and zinc (Zn)] [1]. Zn has been shown to be essential to the structure and function of a large number of macromolecules and more than 300 enzymes [2]. The metal has both catalytic and structural roles in enzymes, while in zinc finger motifs it provides a scaffold organizing protein sub-domains for interaction with either DNA or other protein [3]. Zn is present at high concentration in human seminal plasma; the mean concentration is 2 mM, 100 times higher than in serum [4]. The metal has an important role in testes development, sperm physiologic functions, and Zn deficiency causes hypogonadism [4]. A wide range of Zn

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concentrations is present in the seminal plasma of mammals, ranging from high values for boar (17.7 mg/dL) to low concentrations in fox (1.3 mg/dL) [5]. The most common techniques to collect cat semen are artificial vagina (AV) [6,7] and electroejaculation (EE) [8,9]. Recently, a new technique of urethral catheterization after pharmacological administration (UrCaPI) was described [10]. All techniques yield a good ejaculate containing spermatozoa and seminal plasma. Sperm collection with artificial vagina is an inexpensive technique which does not require physical or chemical restraint of animals, even though a teaser queen and trained tomcat are usually necessary. Electroejaculation can be performed on any male cat that can be safely anesthetized, but it requires specific equipment and is not permitted in all countries. The UrCaPI technique uses an urethral catheter to collect sperm released in the urethra in response to medetomidine administration. The UrCaPI semen samples are characterized by lower total volume and higher spermatozoa concentration (106/ml) compared with EE semen samples, but the total number of spermatozoa (106/ejaculate) is not significantly different between UrCaPI and EE semen samples [10]. The veterinary literature contains only one study on the biochemical characterization of cat seminal plasma because of the difficulty in collecting semen and the small semen volume in this species [1]. In addition, cat has been used as a model for human and nondomestic felid pathologies [11,12]. The study of seminal plasma composition is important because seminal plasma protein profile has been correlated in bovine to semen fertility [13] and freezability [14], and because studies on canine seminal plasma reported that it contains prostate and epididymidal markers [15,16]. Seminal plasma has been analyzed mainly in dog, bull, stallion and ram [13–15,17–19]. The presence of particular proteins has been associated with specific semen parameters in bulls: protein fertility-markers were detected in Holstein bulls using 2D-page [13] and differences in seminal plasma proteins were determined between bulls with high and low semen freezability [14]. A recent study reported that ram seminal plasma obtained using EE or AV did not show any significant difference in total protein content, but with EE the 2D-page protein maps displayed two additional protein spots (15 kDa, 22 kDa) and the loss of one protein (25 kDa) [19]. However the pre- and post-thaw sperm quality was not influenced by sperm collection techniques in ram [20]. SDS-page in canine seminal plasma disclosed specific protein bands as markers of prostate gland and

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epididymidal secretion [15,16]. The B20 band (15.6 kDa), present in high concentration in samples collected from all dogs, was identified as an arginine esterase subunit, considered a marker of normal prostate gland activity [15]. Moreover, bands B9 (42.6 kDa) and B13 (29.2 kDa) were detected as epididymidal secretion markers because of their absence in semen ejaculate samples obtained post-vasectomy [16]. Moreover, as observed in bulls [13], there was a positive correlation in dogs between semen parameters, such as sperm motility and vigor, and specific protein bands (67 kDa, 58.6 kDa) [15]. In the light of a recent report demonstrating differences in semen collected by EE or UrCaPI [10], the present study aimed to separate the proteins in cat seminal plasma using SDS-page and correlate the results to semen parameters. In addition we evaluated whether sperm collection techniques (EE versus UrCaPI) affect the protein profile and protein (g/dL; total amount/ejaculate) and zinc (mg/ dL; total amount/ejaculate) concentrations of semen samples. 2. Material and methods All the chemicals and reagents in this study were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. 2.1. Animals Twenty-one clinically healthy, adult (age range, 1–3 years) mixed-breed male cats were enrolled in this experiment, completed from January to June 2008. The cats, privately owned or belonging to a cat pound, were brought to the Obstetrics and Gynaecology Section of the Clinical Veterinary Department to be submitted to orchiectomy. Nineteen cats had two palpably normal descended testes but two cats were cryptorchid (one bilateral inguinal and the other unilateral inguinal); these two were also included in the study. The experiment was approved by Ethical-Scientific Committee of Alma Mater Studiorum—University of Bologna. 2.2. Semen collection and evaluation A single ejaculate was obtained from five cats by EE and from 12 cats using UrCaPI while 16 samples were collected from four cats using both techniques alternately, once every four days. The four cats submitted to multiple collections were housed individually in boxes under natural photoperiod, with free access to food and water. For semen collection by EE or UrCaPI, the

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animals were injected intramuscularly with medetomidine (Domitor vet®-Pfizer S.r.l., Roma-Latina, Italy; 130 –140 ␮g/kg of body weight) [21]. The EE was performed following the protocol described by Howard et al [9]. In brief, the cat received a total of 80 stimuli divided into three sets (30, 30, and 20) with two to three minutes of rest between sets. Urethral catheterization was performed as described by Zambelli et al [10]. The semen was collected by a 3 French urinary tomcat catheter (Portex® Jackson Cat Catheter) after medetomidine induced spermatozoa release into the urethra. After semen collection, the anaesthesia was maintained with 1.5–2% isoflurane (Isoflo®; Esteve Farma Lda) in oxygen in order to perform orchiectomy. Semen was collected in a pre-warmed plastic 1.5 mL eppendorf tube and the motility, morphology and viability were immediately evaluated. Two ␮l of sperm were diluted with 18 ␮L of Tris-glucose-citrate for accurate determination. The volume was estimated using a variable volume calibrated pipette. A phase-contrast microscope equipped with a warming plate was used to determine the percentage of motile spermatozoa (0 –100%) and the forward progressive motility (score of 0 –5) at ⫻400 magnification, and also to study spermatozoa morphology at ⫻1000 magnification. The sperm concentration was measured using a Bürker chamber. Then semen was centrifuged at 300 ⫻ g for 8 min, the supernatant seminal plasma aspirated without disturbing the sperm pellet, and recentrifuged at 3000 ⫻ g for 15 min. The aliquots of seminal plasma obtained were stored at ⫺80 °C. Sperm pellet was diluted with egg yolk-Tris-glucose-citrate and frozen following the method reported by Zambelli et al [22] to be stored for subsequent studies. 2.3. Protein determination Soluble protein concentration in seminal plasma was measured according to Lowry et al (1951) [23] using a colorimetric assay, the Dc Protein Assay kit (Bio-Rad, Hercules, CA, USA). A standard curve was prepared using 3 dilutions of bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) from 0.5 mg/ml to 1.5 mg/ml. Each sample was tested at 750 nm in triplicate using 96-well microliter plates in a MultisKan EX spectrophotometer. 2.4. Zn analysis Samples of seminal plasma were diluted 1:10 with metal-free distilled water (Merk) and directly aspirated into the flame of an atomic absorption spectrophotometer (Instrumentation Laboratory, Model IL11, equipped with

a deuterium lamp background correction). The instrumental wavelength for Zn was 213.9 and 324.7 for Cu. Metal concentration was reported in mg/dL of seminal plasma. The detection limit (or LOD, limit of detection) was established by analysis of 10 blank solutions and was calculated as 2* ␴ (standard deviation of the blanks). Blank values were 0.010 ⫾ 0.002 ␮g/ml for Zn and 0.006 ⫾ 0.003 ␮g/ml for Cu; hence the detection limits were 4 ng/ml for Zn and 6 ng/ml for Cu. Trace metal standards were run every 20 samples. The accuracy of the method was evaluated by calibration vs. an international standard (CE195: bovine blood-ERM). The concentrations obtained with the method used in this study fell within the confidence interval given by the Community Bureau of Reference. To rule out contamination in UrCaPI samples, we evaluated the zinc concentration of a neutral solution after flushing of the catheter as generally performed for the UrCaPI. 2.5. Gelfiltration chromatography A volume of 0.250 ml seminal plasma was applied onto a Sephadex G-50 chromatography column (0.5 ⫻ 0.20 cm) calibrated with ferritin, albumin and cytochrome c (Sigma) as protein markers, and eluted with Tris-HCl buffer, pH 7.6. The collected fractions were analysed for Cu and Zn by direct aspiration of the solution into the flame of the atomic absorption spectrophotometer as previously described. Absorbance at 280 nm was also measured in a uv-vis spectrophotometer (Beckman DU 530). 2.6. SDS polyacrylamide denaturing gel electrophoresis (SDS-PAGE) Thirty-three ejaculates (five by EE, 12 by UrCaPI and 16 from four cats using both techniques alternately) were collected, but only 23 ejaculates were used for SDS-page because the small sperm volume obtained prevented biochemical analysis. The previous articles about cat seminal plasma analyze only few parameters or perform analysis needing a little amount of semen; instead for the complete biochemical analysis (protein determination, Zn analysis, gel filtration chromatography, SDS-page electrophoresis) at least 150 –200 ␮l of diluted seminal plasma from each sample are needed. In this study, only 15 seminal plasma SDS-page protein profiles were considered valid (seven obtained using sperm collected by UrCaPI and eight using sperm collected by EE) because the others were discharged due to extended protein degradation. Protein and zinc con-

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Table 1 Evaluation of fresh cat sperm collected by urethral catheterization after pharmacological induction (UrCaPI) and electroejaculation (EE). Semen

Volume (␮l)

TM (%)

FPM

Concentration (⫻ 106 /mL)

Total number of spermatozoa (⫻ 106)

UrCaPI EE

20.9 ⫾ 15.1a 89 ⫾ 54 b

59.5 ⫾ 21.1 66.9 ⫾ 24.1

3.5 ⫾ 1.3 4.1 ⫾ 1.4

1453.3 ⫾ 1454.9a 223.8 ⫾ 163.8b

30.3 ⫾ 42.4 19.9 ⫾ 17.6

For each column a vs. b is significantly different [Student t-test; P ⬍ 0.01 or Wald-Wolfowitz runs test for non-parametric data (FPM); P ⬍ 0.01]. TM, total motility; FPM, forward progressive motility.

centrations were then determined only in samples with enough seminal plasma left. Each of the available 15 aliquots of seminal plasma, stored at ⫺80 °C, was normalized for a protein content of 20 ␮g before loading on a 12% Bis-Tris gel under reducing conditions. Seven UrCaPI and eight EE samples were analysed; each sample of seminal plasma was run in at least in two different gels. SDS-polyacrylamide gel electrophoresis (PAGE) was performed in an Xcell SureLock Mini-Cell with MES (Invitrogen Italia S.R.L.—San Giuliano Milanese, MI Italia) running buffer at pH 7.3. Each gel was also loaded with standard proteins of known molecular weight (See Blue-Invitrogen Italia S.R.L.—San Giuliano Milanese, MI Italia). The electrophoresis system was connected to a power supply (Power Pack Basic— Bio-Rad, Hercules, CA USA) with constant voltage of 200 V for 40 min. Gels were stained with Silver Quest (Silver staining kit- Invitrogen) following the standard protocol. Gels were digitised and band optical densities were quantified using ImageJ 1.31v software (http://rsb.info. nih.gov/ij/). Each band was therefore converted into a densitometry trace allowing calculations of integrated optical density (IOD). 2.7. Statistical analysis Data from semen evaluation are expressed as mean ⫾ S.D. and were analysed using a t-test for independent samples or a Wald-Wolfowitz runs test, depending on data distribution. In particular the forward progressive motility (FPM) was analysed using a Wald-Wolfowitz runs test for non-parametric data. Data from protein

concentration (g/dL; total protein amount/ejaculate), zinc concentration (mg/dL; total Zn amount/ejaculate) and IOD are reported as mean ⫾ S.D. and were analysed using a t-test. The correlation between zinc and protein concentration was determined using a Pearson test for positive correlation. All tests were elaborated applying a software package (Statistica for Windows, Stat Soft Inc., Tulsa, Oklahoma, USA). A value of P ⬍ 0.01 was considered significant in Table 1, whereas a value of P ⬍ 0.05 was considered significant in Table 2. 3. Results 3.1. Semen quality Semen characteristics are listed in Table 1. The semen collected by UrCaPI was characterized by lower volume (P ⬍ 0.01) and higher sperm concentration (P ⬍ 0.01), whereas total motility (TM), forward progressive motility (FPM) and total number of spermatozoa were similar (P ⬎ 0.01) in samples collected using EE or CT. 3.2. Zinc and protein concentration in seminal plasma Data on Zn concentration (mg/dL; Total Zn/ejaculate) and protein concentration (g/dL; Total protein/ ejaculate) are reported in Table 2. Zn concentration varied from 1.1 to 2.0 mg/dL in UrCaPI samples and from 0.27 to 0.84 mg/dL in EE samples. Among all specimens examined Zn (mg/dL) and protein (g/dL) were significantly less concentrated in EE samples (P ⬍ 0.01); while total Zn and protein amount/ejaculate were

Table 2 Zinc and total protein concentration in cat seminal plasma collected by urethral catheterization after pharmacological induction (UrCaPI) and electroejaculation (EE).

UrCaPI EE

[Zn] mg/dL

Total proteins g/dL

Total proteins/ejaculate

[Zn]/ejaculate

1.56 ⫾ 0.45a (n ⫽ 4) 0.56 ⫾ 0.21b (n ⫽ 7)

1.09 ⫾ 0.22a (n ⫽ 7) 0.31 ⫾ 0.06b (n ⫽ 8)

184.7 ⫾ 17.5 (n ⫽ 7) 208.4 ⫾ 42.4 (n ⫽ 8)

258.7 ⫾ 37.8 (n ⫽ 4) 432.3 ⫾ 103.2 (n ⫽ 7)

For each column a vs. b is significantly different (Student t-test; P ⬍ 0.05).

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not significantly different (P ⬍ 0.05) in sample collected by UrCaPI or EE. A positive correlation between zinc and protein concentration was found (r ⫽ 0.953, P ⬍ 0.01). Seminal plasma proteins were subjected to fractionation by gel-filtration chromatography to separate them according to their molecular weight. On the resulting fractions the absorbance at 280 nm was indicative of protein presence, while Zn concentration was measured by atomic absorption spectrofotometry. A gel-filtration profile for seminal plasma obtained by EE is shown in Fig. 1. Zinc co-eluted with the two main protein peaks, namely bound to high molecular weight ligands (fractions 4 –7) and intermediate molecular weight ligands (fractions 9 –12); a small peak was also present bound to low molecular weight ligands, presumably peptides or free aminoacids (fractions 16 –18). 3.3. Separation of seminal plasma proteins by gel electrophoresis and comparison between UrCaPI and EE protein patterns Proteins in seminal plasma were separated by SDSPAGE. As an example a gel image is reported in Figure 2. The integrated optical density of protein bands is reported in Figure 3. More than 30 protein bands were found, even though they were not always present together in the same sample. Apparent molecular weight (MW) ranged from 3.5 to 200 kDa. Major protein bands were concentrated in two different zones of the electrophoretic pattern: high MW proteins between 60 and 200 kDa and low MW proteins less than 22 kDa. Eight proteins bands, namely bands at 5.5, 12, 22, 28, 66, 70,

Fig. 1. Gel filtration profile of cat seminal plasma obtained by EE. Seminal plasma proteins were subjected to fractionation by gelfiltration chromatography to separate them according to their molecular weight. On the resulting fractions the absorbance at 280 nm was indicative of protein presence, while Zn and Cu concentrations were measured by atomic absorption spectrofotometry. The arrows indicate the three peaks of molecules binding Zn.

Fig. 2. Denaturing SDS-PAGE electrophoresis profile of cat seminal plasma. Lane 1 molecular weight marker (200-3 kDa); lanes 2–5 samples collected by UrCaPI; lanes 6 –9 samples collected by EE.

80 and 200 kDa were present in all the subjects analysed. The 5.5 and 12 kDa bands were more evident than the others, especially in UrCaPI samples. As an example, Figure 4 shows the electrophoretic profiles of seminal plasma sampled from the same cat using UrCaPI and EE. In general, more protein bands are present in the UrCaPI samples, in particular P55 and P14 are present exclusively in UrCaPI samples (Figure 4). In general, three proteins, P200, P80, P28 are significantly more concentrated in EE samples, while P66, P22, P12 and P5.5 are more concentrated in UrCaPI samples (Figure 4). 4. Discussion The values of Zn and protein we found in cat seminal plasma are lower than those reported for other animals. In addition, contrary to what happens in ram

Fig. 3. Integrated optical density (I.O.D.) profile of cat seminal plasma proteins separated by SDS-PAGE. Grey bars: samples obtained by UrCaPI (n ⫽ 7); white bars: samples obtained by EE (n ⫽ 8). Data are reported as means ⫾ SD. For each protein significant differences between UrCaPI and EE samples have been calculated with Student t-test and is indicated by an asterisk (*) (P ⬍ 0.05).

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Fig. 4. Denaturing SDS-PAGE electrophoresis profile of seminal plasma obtained from the same cat by urethral catheterization (UrCaPI) and electroejaculation (EE). In the figure, P55 and P14 protein bands are indicated by two rectangles.

[19], the collection technique influenced the concentrations of zinc (mg/dL) and protein (g/dL) that were significantly higher in samples collected by UrCaPI than those collected by EE. Instead the total Zn and protein amount/ejaculate are not significantly different (P ⬍ 0.05) in sample collected by UrCaPI or EE (Table 2). A previous study [10] states that the spermatozoa concentration (⫻ 106/ml) is higher in UrCaPI samples respect to EE samples, but the total number of spermatozoa (⫻ 106) is the same in UrCaPI and EE ejaculates; therefore we suppose that zinc and proteins have a similar behaviour to spermatozoa. Most of the bands in cat seminal plasma were present at high molecular weight between 200 and 60 kDa (n ⫽ 5) and at low molecular weight, less than 22 kDa (n ⫽ 7). The number of protein bands also exceeds 30 in dog, but in this species most proteins had a molecular weight below 17 kDa, with the 15.6 kDa protein present at high concentrations in all dogs examined [15,16]. The authors hypothesized that this protein could be a subunit of arginine esterase, which is present at high concentrations in canine prostatic secretions and is considered a specific immunological marker to assess the normality of prostate gland, similarly to human PSA [24]. Instead, bull and ram present fewer protein bands [14,19] concentrated in a zone of the electrophoretic pattern between 11–14 kDa and 24 –26 kDa [14,19]. The number of protein bands in stallion is similar to the protein bands in bull but ranges from 14 kDa to 120 kDa [18]. The P66 protein is present in all ejaculates and has the same molecular weight as serum albumin, the most abundant plasma protein. P66 has also been found in human seminal plasma [25] and is though to act as a sink for cholesterol, which is removed from the sperm membrane during capacitation [26]. The samples col-

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lected by UrCaPI presented more protein bands than those collected by EE. In particular, comparing the protein profile of the seminal plasma collected from the same cat using UrCaPI and EE, UrCaPI disclosed two protein bands not present in ejaculates collected by EE (P55, P14) (Figure 4). Moreover P66, P22, P12, and P5.5 were more concentrated in UrCaPI samples than those collected by EE. Performing UrCaPI, the catheter brushes the urethral mucosae causing microtraumas and collecting urethral cells at the same time. However, no cat presented clinical signs the days after sperm catheter collection and no ejaculate showed red blood cells macroscopically. Therefore the higher concentration of these proteins in UrCaPI samples could partially derive from the microtrauma caused by the recovery method, or these proteins could not have a prostatic or bulbourethral origin and hence present a low concentration in EE samples below the SDS-page detection limit. Further studies are necessary to confirm these suspicions. On the other hand, three proteins (P200, P80, P28) were more abundant in the EE samples: these proteins could be related mainly to prostatic or bulbourethral origin. A recent study [27] used 2D-gel electrophoresis to identify major secreted proteins from mouse prostate, including spermine-binding protein, serine protease inhibitor, peroxiredoxin-6, glucose-regulated protein 78 and zinc-␣2-glycoprotein. One of these proteins, namely glucose-regulated protein 78, has a molecular weight of 78 kDa, which could correspond to the P80 protein identified in our study. Finally, there was no relationship between protein profile and semen parameters as reported in other species [15]. The semen collected from the two inguinal cryptorchid cat lacked spermatozoa but presented a seminal plasma protein profile similar to those of the other cats. Also if the limited number of cryptorchid cats does not permit us to perform a statistical analysis, it is possible to suppose that cryptorchidism compromises the spermatozoa production of the testis, but does not modify seminal plasma secretion produced by the sexual accessory glands. In fact, both cryptorchid cats presented penile spines, implying testosterone production and consequently the correct development of the sexual accessory glands. In summary, semen collection by EE is related to the absence of some proteins in seminal plasma (P55 and P14), and alters three protein bands (P200, P80, P28). However, considering the results of a recent study [10], it is reasonable to claim that although the ejaculates collected by UrCaPI or EE present some protein profile differences, they might have the same fertilizing ability.

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