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Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity
Theriogenology 82 (2014) 1273–1280
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Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity _ n ski*, Ma1gorzata Ochota, Sylwia Prochowska, Wojciech Niza Agnieszka Partyka Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 19 August 2014 Accepted 19 August 2014
This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 106/mL, 5 106/mL, and 1 106/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 106/mL dilution rate, spermatozoa diluted to 1 106 sperm/mL had a significantly lower proportion of motile (31.1% 19.8 and 0.7% 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% 3.1 and 69.1% 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% 11.9 vs. 75.9% 10.6) and chromatin status (defragmentation index, 3.3% 0.97 vs. 3.4% 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cat semen Sperm concentration Dilution effect Flow cytometry
1. Introduction The handling of semen and its processing associated with assisted reproductive technologies (ART) very often includes extension of sperm, as in the case of preparing spermatozoa for cryopreservation, IVF, flow cytometric assessment, or cell sorting by chromosomal sex. The latter two require high dilution, which may be detrimental for the motility and viability of spermatozoa, as is the case in bovine [1,2] and ram semen [3]. This phenomenon has been described as the “dilution effect” [4]. It is thought that the dilution effect is connected with a partial or even complete removal of some essential seminal
* Corresponding author. Tel.: þ48 71 3205315; fax: þ48 71 3205306. _ n ski). E-mail address: [email protected] (W. Niza 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.08.012
plasma (SP) compounds that protect the sperm from damage, thereby contributing to the deleterious effects of semen dilution. Although the role of SP is ambiguous and many authors reported both its beneficial and detrimental influence on sperm characteristics [5–7], the presence of SP seems to play a crucial role in the case of excessive semen dilution. The evidence in support of this hypothesis is that the re-addition of SP or its compounds to the highly diluted sperm can prevent or reduce its damage in sheep [3,8,9], rabbits [5], cattle [2,9], and pigs [9–12]. Articles dedicated to feline sperm dilution are scarce. Howard et al. [13] showed that even a twofold dilution of electroejaculated feline semen resulted in a decrease of sperm motility. To the authors’ knowledge, no other information on this topic is available, although the semen in this species is characterized by a high concentration of sperm cells and an extremely low volume of ejaculate
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ranging from 10 mL [14] to 200 mL [15]. Therefore, high dilution rates of semen are usually necessary before sperm cell assessment, especially in semen collected by urethral catheterization [14]. The dilution of semen is also necessary before artificial inseminationdinsemination dose for intrauterine insemination of queens varies from 4.2 106 to 10 106 of sperm cells deposited in 30 mL [16] up to 200 mL [17,18]. Flow cytometry, which is widely used in andrology laboratories, is a valuable tool for the assessment of semen [19]. One of the advantages of flow cytometry is the possibility to assess a large number (>10,000) of spermatozoa in a short period of time. However, it requires a relatively large sample volume. This problem is solved by sample dilution. In cats, the assessment of semen by flow cytometry is rarely used most likely because of a low ejaculate volume and because a small overall number of spermatozoa can be obtained. Depending on the collection method, the total sperm count varies from about 20 106 in the case of electroejaculation [20] and urethral catheterization [14] to 80.1 106 in the case of an artificial vagina [21]. Such a small number of spermatozoa per ejaculate is a limiting factor with reference to the parallel assessment of many sperm characteristics or to combine flow cytometry sperm evaluation with other procedures (e.g., cryopreservation or artificial insemination). The possibility of assessing highly diluted samples (1 106 cells/mL, as recommended by manufacturers of flow cytometers and fluorescent dyes) through flow cytometry may enable a simultaneous broad sperm evaluation and its use for ART. It could also widen the spectrum of the assessed sperm parameters. However, due to the fact that extreme dilution may influence semen characteristics, there is a theoretical risk of an artifactual change of the results of the semen evaluation. Consequently, the laboratory assessment of the fertilizing potential of a particular male may be erroneous. Urethral catheterization is a novel method of semen collection in cats, easy to introduce in everyday practice. In comparison with ejaculated spermatozoa (obtained via an artificial vagina or electroejaculation), semen collected using this method is characterized by a lower volume, higher sperm concentration, and a minimal presence of SP [14]. Thus, it very often requires a high dilution, but it has not been checked if such extensions may affect the sperm characteristics. Therefore, the aim of this study was to assess the influence of high sample dilution on spermatozoal motility, viability, acrosome integrity, and chromatin status in the feline urethral semen. 2. Materials and methods 2.1. Animals Spermatozoa were collected from eight privately owned male cats scheduled for routine castration procedures at the Department of Reproduction and Farm Animal Clinic of the Wroclaw University of Environmental and Life Sciences (Poland). All tomcats were clinically healthy Domestic Shorthair cats, aged between 8 and 36 months. All procedures were performed with the consent of the Second Local Ethical Committee in Wroclaw.
2.2. Semen collection Tomcats presented for routine orchidectomy were anesthetized using medetomidine hydrochloride im 80 mg/ kg of body weight (Sedator 1.0 mg/mL, Novartis, Poland) combined with ketamine im 5 mg/kg of body weight (VetKetam 100 mg/mL, VetAgro, Poland). To reduce the postoperative pain, an injection of meloxicam sc 0.3 mg/kg of body weight (Metacam 5 mg/mL, Boehringer Ingelheim Vetmedica, Germany) was given before the anesthesia. After the orchidectomy, the cats were given a mixture of benzathine benzylpenicillin 100,000 IU/mL, procaine benzylpenicillin 100,000 IU/mL, and dihydrostreptomycin sulfate 200 mg/mL im 1 mL/10 kg of body weight (Shotapen L.A., Virbac, France). The urethral semen was collected as previously described by Zambelli et al. [14]. Briefly, a tomcat urinary catheter with its tip cut to get a shorter, open-ended catheter was inserted approximately 9 cm into the urethra, taking care not to reach the bladder. Subsequently, the catheter was removed from the urethra for the purpose of collecting a semen sample. Immediately after successful sample collection, it was placed in a prewarmed Eppendorf tube containing 200 mL of semen extender based on TRIS buffer ((3.02%, wt/vol) TRIS (Sigma–Aldrich, Poland), 1.35% (wt/vol) citric acid (Sigma–Aldrich), 1.25% (wt/vol) fructose (Sigma–Aldrich), in bi-distilled water; pH 6.5). 2.3. Experimental design and semen evaluation Each sperm sample was assessed for sperm motility and sperm concentration immediately after collection. The sample was then divided into three aliquots and a semen extender was added to reach the final concentration of spermatozoa 10 106/mL, 5 106/mL, and 1 106/mL. The 1 106/mL dilution rate was chosen as recommended by manufacturers of flow cytometers and fluorescent dyes. The 10 106/mL was the highest possible concentration to prepare, according to the amount of sperm obtainable from a cat that allows the creation of three dilutions for comparison. The concentration 5 106/mL was the value in between. Immediately after dilution, each sample was assessed for sperm motility and then prepared for a flow cytometric evaluation of viability, acrosome integrity, and chromatin status. 2.3.1. Sperm motility and concentration In order to assess the motility, 10 mL of a sperm sample (raw semen and a sample from each of the investigated dilutions) was placed on a prewarmed slide and the percentage of motile sperm was subjectively estimated under a contrastphase microscope at a 200 magnification by three independent researchers and the mean value was calculated. To evaluate sperm concentration, a 10-mL aliquot of the semen sample was diluted in 200 mL of distilled water and cells were counted in 80 squares of the Thoma chamber. Counting was repeated in a second chamber and the mean value was calculated. 2.3.2. Flow cytometry assessment Measurements were carried out on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. The fluorescent probes were excited by an Argon ion 488 nm laser. Detection
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of green fluorescence was set with an FL1 band-pass filter (530 nm ⁄ 30 nm) and red fluorescence was measured using an FL2 longpass filter (>670 nm). Green florescence of acridine orange (double-stranded DNA) was detected on the FL1 detector and red fluorescence of acridine orange (AO; singlestranded DNA) was identified on the FL3 detector. Acquisitions were measured using the CellQuest 3.3 software (Becton Dickinson). The non-sperm events were gated out based on scatter properties and were not analyzed. A total of 20,000 events were analyzed for each sample. 2.3.2.1. Viability. The viability was assessed using the dual fluorescence staining with SYBR-14 and propidium iodide (PI) as described by Pukazhenthi et al. [15], with minor changes. Spermatozoa were stained using the sperm viability kit (Live/Dead Sperm Viability Kit, Life Technologies Ltd, Carlsbad, CA, USA). An aliquot of 300 mL of diluted samples was pipetted into cytometric tubes, and 2.5 mL of SYBR-14 working solution was added. The working solution was obtained by diluting a commercial solution of SYBR-14 in distilled water with a ratio of 1:49. Samples were mixed and incubated at room temperature in the dark for 10 minutes. Then, the cells were counterstained with 1.5 mL of PI 3 minutes before analysis. Four subpopulations of cells were observed on dot plots of PI/SYBR-14 fluorescence: dead spermatozoa (PIþ/SYBR14), moribund spermatozoa (PIþ/SYBR-14þ), live spermatozoa (PI/SYBR-14þ), and a nonlabeled population (PI/SYBR-14) that was considered to be debris [22]. 2.3.2.2. Acrosome integrity. The acrosome damage was assessed using lectin PNA from Arachis hypogaea Alexa Fluor 488 Conjugate (Life Technologies Ltd.). Because a protocol for cats has not yet been described, we used a modified protocol used in our laboratory for fowl semen [22]. Briefly, 5 mL of PNA working solution (1 mg/mL) was added to 500 mL of semen samples and incubated for 5 minutes at room temperature in the dark. Following incubation, the supernatant was removed by centrifugation (500 g for 3 minutes), and the sperm pellets were resuspended in 500 mL of TRIS buffer. For counterstaining, 1.5 mL of PI was added to samples before cytometric analysis. Dot plots of PNA/PI-stained spermatozoa showed four populations of cells: live cells with an intact acrosome (PI/ PNA), live cells with a ruptured acrosome (PI/PNAþ), dead cells with an intact acrosome (PIþ/PNA), and dead cells with a ruptured acrosome (PIþ/PNAþ) [22]. 2.3.2.3. Chromatin status. The AO stain was used to assess sperm DNA integrity as previously described in cats [23], with minor changes. The suspension (100 mL) was subjected to brief acid denaturation by mixing with 200 mL of a lysis solution (Triton X-100 0.1% (vol/vol), NaCl 0.15 M, HCl 0.08 M, pH 1.4), held for 30 seconds, and mixed with 600 mL of an AO solution (Life Technologies Ltd) (6 mg AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6). After 3 minutes, the samples were aspirated into a flow cytometer. The main population that represents spermatozoa with a predominantly normal double-stranded configuration of DNA was observed on dot plots. Sperm cells located to the
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right of this main population represent cells, which show an increased amount of red fluorescence, indicating denatured DNA (DFI). Spermatozoa above the upper border of the main cluster of the sperm population represent spermatozoa that have an abnormally high DNA stainability [22]. 2.4. Data analysis The results obtained are presented as mean SD of measurements. Data were analyzed using ANOVA and Duncan’s multiple range test. The level of significance was set at P value less than 0.05. An elaboration of tests was carried out using the Statistica software for Windows, StatSoft Polska Sp. z o.o. 3. Results 3.1. Sperm motility The motility of the raw semen was 66.9% 20.2. Sperm motility was reduced by increasing sperm dilution. Samples diluted to the concentration of 10 106/mL showed a motility decrease by at least 50% (average motility, 31.1% 19.8), which was further exacerbated in a concentration of 5 106/mL (average motility, 16.7% 18.5). In the most diluted samples (1 106/mL), single motile spermatozoa were observed (average motility 0.7% 1.6) or none at all. The results are presented in Figure 1. In many samples with a concentration level of 10 106/ mL and 5 106/mL, the spermatozoa showed no progressive movement. Only a subtle oscillation was observed, which could indicate an adherence of the motile spermatozoa to the glass surface. The magnitude of this phenomenon differed between samples. It was seen less frequently in specimens of high motility than those characterized by low motility, where this oscillation was often the only type of movement observed. 3.2. Flow cytometry assessment The results of viability, acrosome integrity, and chromatin status are shown in Table 1. The percentage of live cells (SYBRþ/PI) showed a statistically significant difference (P ¼ 0.016) between the 5 106/mL and 1 106/mL dilution rates and highly significant differences (P ¼ 0.009) between the 10 106/mL and 1 106/mL dilution rates. Simultaneously, the percentage of dead cells (SYBR/PIþ) differed significantly (P ¼ 0.02) between the 10 106/mL and 1 106/mL (6.5 3.2 vs. 21.3 11.9) dilution rates. There were no significant differences in terms of the acrosome integrity and chromatin status between different dilution rates in the assessed populations of spermatozoa. 4. Discussion The quality of cat urethral semen in our study was comparable to the results obtained by other authors in terms of motility (66.9% 20.1 in comparison with 78.1% 9.6 [14] and 50% 12.4 [20] and 50.4% 20.3 [24]) and viability (88.3% 3.1 compared with 82.2% 18.4 [24]
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Fig. 1. Motility of spermatozoa in different concentration levels.
a.b.c
Different superscripts within bars indicate significant differences, P < 0.05 (N ¼ 8).
and 80.0% 10.1 [14]). In our study, the results of the acrosome integrity were higher than those obtained by Filliers et al. [24], who used a fluorescent microscope (>90% vs. 84.2% 6.8, respectively). This difference may be caused by the type of the extender used and the differences in the anesthetic procedures, sperm preparation protocols, and evaluation methods [20]. This was the first study analyzing cat urethral semen in terms of chromatin integrity. Our findings obtained with the use of flow cytometry (normal DNA stainability, 93.4% 2.9) are similar to the results obtained by other authors with the use of AO staining and a fluorescent microscope for epididymal (95.2% 2.6) [25] and ejaculated (97.1% 2.1) [26] sperm cells. In the present study, we confirmed a deleterious effect of increasing dilution on sperm motility and viability that was previously described by many authors with reference to ejaculated bull [1,2,8], boar [10–12], and ram semen [3,8]. However, the underlying mechanism of this phenomenon has not yet been fully explained. In general, it is thought that the dilution of semen presumably removes adsorbed (“coating”) proteins, natural antioxidants, and other beneficial components in SP required for the maintenance of the membrane integrity and function of spermatozoa [27]. However, it seems that the mechanisms and factors responsible for the “dilution effect” are different when it comes to the loss of motility and loss of viability. In our study, the loss of motility induced by dilution was much more marked than the loss of viabilitydthe percentage of viable cells in the highest dilution was on average 69%, whereas the number of motile spermatozoa in this group decreased to 0.7%. Many other authors noticed that the viability and motility do not always correlatedsometimes viable spermatozoa were assessed as immotile and motile
ones were classified as nonviable [8,9,12]. Therefore, these two parameters should be interpreted separately. Factors like temperature, pH, and osmolality, either below or above physiological ranges, can impair spermatozoal motility [4,15], but as the negative effect of these factors is widely known, they will not be discussed here. Apart from these factors, proteins are thought to be essential for maintaining sperm motility. An evidence to support this hypothesis is that the deleterious effect of salt diluents on spermatozoa can be at least partly overcome by the inclusion of certain organic substances, mainly of a colloidal nature [4]. Moreover, the loss of motility in SPsubtracted samples can be restored by adding BSA [28,29]. In addition, the phenomenon of spermatozoa sticking to the glass surface, which was observed in our study as well as by other authors [6,29], may indicate that the dilution alters the sperm membrane surface proteins. Removal of these proteins creates charge differences, which promote agglutination [9]. Another explanation for the loss of motility in the diluted samples is a low level of motilitystimulating factors [29]. However, because semen extenders are nowadays supplemented with energy sources (glucose and fructose), citric acid, and many other constituents, the lack of these factors may be limited to cases where only simple diluents (e.g., normal saline) are used. Complex extenders can maintain spermatozoal motility equally well or even better than SP [30]. The matter is more intricate in the case of sperm viability. Ashworth et al. [3] reported that the survival of ram sperm in high dilution was improved by the addition of low molecular weight components such as sucrose, lactate, pyruvate, ethylene glycol tetraacetic acid (EGTA), Ca2þ, Mg2þ, and phosphate. Similarly, in the study of Catt et al. [8], a complex
Table 1 Plasma membrane integrity, acrosome integrity, and chromatin status of feline urethral spermatozoa in three dilution rates. Spermatozoa population
Live (SYBRþ/PI) Live with an intact acrosome (PI/PNA) Defragmentation index (%DFI) a,b
Concentration 10 106/mL
5 106/mL
1 106/mL
88.3 3.1a 76.7 11.9 3.3 0.97
85.9 4.0a 79.8 11.0 2.8 1.2
69.1 12.8b 75.9 10.6 3.4 1.7
Different superscripts within rows indicate significant differences, P < 0.05 (N ¼ 8).
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extenderdBeltsville thawing solutiondmaintained the viability of boar and ram spermatozoa, with or without the addition of SP. This suggests that simple metabolites or ionic components may be the most important components of SP for the maintenance of cell viability. Thus, the dilution effect on spermatozoa may be minimized by the use of wellbalanced semen extenders. However, seminal proteins are also important in the case of high semen dilution, as they are thought to exert the most significant response to SP [31]. The content and function of seminal proteins have been intensively studied for last 20 years. As a result, many proteins were identified and described (for review see [32] and [33]). A positive or negative correlation of particular proteins with male fertility [34,35], sperm quality [36–38], and freezability [39,40] has been reported. However, the role of proteins in maintaining spermatozoal viability in high dilution has not, to date, been fully explained. In general, they are considered to stabilize sperm cells by coating the sperm surface [41,42]. These “coating factors” tend to be eluted from spermatozoa on dilution, resulting in destabilization of their plasma membranes leading to their subsequent death [3]. Contrary to motility, the effect is highly specific, linked not with the presence of proteins in general, but with a defined protein. This was proved in the case of ram semen, where the addition of bovine serum albumin (65–70 kDa) did not prevent the loss of viability, instead beneficial agents that protected spermatozoa from the dilution effect were found in 5 to 10 kDa fraction of SP [3]. This subject was studied deeper in boars and it was shown that the addition of an isolated nonheparin–binding spermadhesin PSP-I/PSP-II heterodimer to extremely diluted semen resulted in a greater percentage of viable and motile cells, whereas the addition of heparinbinding spermadhesin (HBP) had a substantial detrimental effect on the survival of spermatozoa [12]. Studies dedicated to SP proteins in cats are limited [20], and, to the authors’ knowledge, no information about its protective or detrimental influence on highly diluted semen is available. Our results suggest that although urethral semen is characterized by a minimal presence of SP [14], probably some plasma compounds are present in urethral semen and play an important role in spermatozoal protection against high dilution. Further studies on sperm and SP proteomics are required to establish the exact agents. Proteins are responsible not only for maintaining sperm motility and membrane integrity but also for sperm capacitation and fertilization of the oocyte (for the review see [43]). It is well known that SP possesses factors that can decapacitate sperm cells and prevent/inhibit the acrosome reaction (reviewed by [44]). This effect is undesirable in case of in-vitro fertilization; therefore, removal of SP is a routine procedure before IVF [45]. On the other hand, both cooling and cryopreservation cause capacitation-likechanges, which may interfere with fertilizing potential of sperm cells. These changes can be reversed or minimized by the addition of SP [46–48]. In our study, higher dilution rates did not cause changes in the acrosome integrity, which is consistent with results obtained by Catt et al. [8] when analyzing fresh ram and boar semen and contrary to the results for cryopreserved bull spermatozoa [49]. The supplementation of SP to ram semen lowered the number
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of acrosome-reacted cells after sex sorting [9], but not after cryopreservation of sex-sorted sperm [50]. These discrepancies in the influence of SP on acrosome status may be a result of different experimental conditions, such as the level of dilution/semen concentration, additional processing of semen (sex –sorting and cryopreservation), methods of evaluating acrosome integrity, species-specific characteristics, and other factors that are discussed below. In our study, dilution did not affect the chromatin structure, which is in agreement with the results for SP removal in dogs [51] and stallions [52]. However, in stallion removal of SP was protective to sperm DNA integrity during 24 and 48 hours cooled-storage [52]. In dogs converselydremoval of SP caused significant increase in %DFI after 3 hours of post-thaw incubation [51]. In humans, a fivefold dilution decreased sperm nuclear resistance to decondensation in SDS [53]. These examples show that semen dilution or SP removal can have an influence on sperm DNA, but the effect depends on other conditions like time and species factor, which are discussed below. It must be emphasized that our study refers only to the high rates of sperm dilution. Moderate dilution of electroejaculated cat semen had no effect on sperm parameters [54,55]. Similar results were obtained by Castellini et al. [5] in rabbits – two- to fivefold SP dilution increased motility parameters, but further dilution caused sperm deterioration. Other studies revealed that the optimal level of SP contents is 10%d a higher concentrations of SP did not further enhance spermatozoal survival [3], had no effect on the viability [6], or even appeared to be detrimental to the sperm motility and membrane integrity [9]. This indicates that SP contains not only beneficial agents but also detrimental ones (like some specific proteins [12], leukocytesda source of ROS [56,57], bacteria [58], hemoglobin [59]), and an excessive amount of SP may have a negative effect to sperm cells. Similarly, it must be taken into consideration that our study examined only a short-time effect of dilutiondup to 30 minutes (the time of the semen assessment). The immediate detrimental effect, which we observed, was also described by other researchers [3,5,11]. This effect is an important factor when assessing fresh sperm. In this case, the role of SP may be considered positive. However, ART requires very often prolonged storage of semen. In those conditions, the influence of SP and its dilution may be different. In vivo spermatozoa have only short-lasting contact with SP, as they quickly swim free to further parts of the female tract. Therefore, a long contact of spermatozoa with SP, as in the case of ART, seems to be unnatural. During the storage period, spermatozoa are exposed to potential detrimental components included in SP for a long time and, simultaneously, protective agents may be “used up.” Hence, the effect of SP dilution/removal/addition during long incubation and cold storage has already been the point of interest of many researches, with various results [28,49,60–64]. In general, the presence of SP was detrimental to sperm cells [61,62,64], although partial removal gave better results than either leaving whole SP or its complete removal [60,62], which is in agreement with the previous paragraph. Another aspect that has to be considered is that fresh semen was assessed in our study. Numerous studies have
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reported a deleterious influence of SP on semen cryopreservation results [7,65–67], particularly in males characterized by poor freezability [68]. Hence, SP is usually removed before freezing [69]. Studies involving post-thaw semen dilution have been contradictory. For example, in rams, post-thaw dilution decreased the viability and motility of spermatozoa [66]. When SP was removed before freezing, post-thaw dilution resulted in better semen quality in cats [70] and dogs [71]. In cats, resuspension of cryopreserved epididymal spermatozoa in SP gave worse results than resuspension in TRIS [72]. On the one hand, semen extension after freezing– thawing can dilute detrimental factors like ROS, hemoglobin, and metabolites accumulated during the freezing procedure and is consequently considered to be beneficial. On the other hand, it was shown that SP can restore the membrane damaged during cryopreservation [73]. In addition, the type of semen (e.g., ejaculated and epididymal) and semen collection method (whole ejaculate and only sperm-rich fraction) can affect the results, as semen obtained using different methods varies in SP content and concentration [20]. Although there are numerous studies comparing different types of semen, none of them (to the authors’ knowledge) refers directly to the high semen dilution. When studying SP addition, results were contradictory. For example, SP addition to epididymal sperm before freezing–thawing had positive effects in the Iberian red deer [74] but did not improve sperm quality in a cat [75]. In the stallion, SP had a stimulatory effect on the motility of epididymal spermatozoa during cooled storage but depressed motility in ejaculated spermatozoa [76]. In this study, we used feline urethral semen. Our observations for epididymal sperm suggest that dilution in a TRIS-buffered semen extender had none or much lesser influence on this kind of semen (Prochowska, unpublished results). We discussed our results for feline semen in reference to other species (due to the lack of articles dedicated to the cat), but one should bear in mind that there are species-specific differences in the susceptibility of spermatozoa to damagedram sperm cells are more sensitive to dilution than boar and bull ones [3,8]. The differences in the composition of SP from different species [77] could explain this and other species-specific differences in terms of the benefits of SP [6,9] and the negative effect of adding heterologous SP [8]. Moreover, semen composition differs not only among species but also among and within individuals [9,35,37,39]. The variability in SP contents can be influenced by the season [36] and the methods of semen collection [20]. From this point of view, the absence, presence, under- or overexpression, and critical concentration of specific proteins in the ejaculate of individual males could alter sperm functions. This may explain the variability of results obtained by different authors for the same species (e.g., [5] and [6] for rabbits; [1] and [49] for bulls), for different males [2,9,10,49] and different ejaculates of the same male [1]. 4.1. Conclusion Feline urethral semen is susceptible to the dilution shockdin highly diluted samples the number of motile and membrane intact cells decreased. This immediate change of viability and motility in highly extended samples suggests
that these features can be easily artifactually changed. Such possibility should be taken into consideration during semen assessment and when comparing results obtained by different authors. Acknowledgments We acknowledge the financial support from MNiSW/ NCN N N308 576540 (N308 576540). The authors are grateful to Iwona Zbyryt (MSc) for her help with the flow cytometric analysis and to Barbara Smalec (MSc) for her excellent technical assistance. References [1] Garner DL, Thomas CA, Allen CH. Effect of semen dilution on bovine sperm viability as determined by dual-DNA staining and flow cytometry. J Androl 1997;18:324–31. [2] Garner DL, Thomas CA, Gravance CG, Marshall CE, DeJarnette JM, Allen CH. Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology 2001;56:31–40. [3] Ashworth PJ, Harrison RA, Miller NG, Plummer JM, Watson PF. Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod Fertil Dev 1994;6:173–80. [4] Mann T. The biochemistry of semen and of the male reproductive tract. London: Methuen and Co.; 1964. [5] Castellini C, Lattaioli P, Moroni M, Minelli A. Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa. Anim Reprod Sci 2000;63:275–82. [6] Dott HM, Harrison RAP, Foster GCA. The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma. J Reprod Fertil 1979;55:113–24. [7] Moore AI, Squires EL, Graham JK. Effect of seminal plasma on the cryopreservation of equine spermatozoa. Theriogenology 2005;63: 2372–81. [8] Catt SL, O’Brien JK, Maxwell WMC, Evans G. Assessment of ram and boar spermatozoa during cell-sorting by flow cytometry. Reprod Domest Anim 1997;32:251–8. [9] Maxwell WM, Welch GR, Johnson LA. Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Dev 1997;8:1165–78. [10] Caballero I, Vázquez JM, Centurión F, Rodríguez-Martínez H, Parrilla I, Cuello C, et al. Comparative effects of autologous and homologous seminal plasma on the viability of largely extended boar spermatozoa. Reprod Domest Anim 2004;39:370–5. [11] Caballero I, Vázquez JM, García EM, Roca J, Martínez EA, Calvete JJ, et al. Immunolocalization and possible functional role of PSP-I PSP-II heterodimer in highly extended boar spermatozoa. J Androl 2006; 27:766–73. [12] Centurion F, Vazquez JM, Calvete JJ, Roca J, Sanz L, Parrilla I, et al. Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution. Biol Reprod 2003;69:640–6. [13] Howard JG, Brown JL, Bush M, Wildt DE. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoa motility and morphology after swim up processing. J Androl 1990;11:204–15. [14] Zambelli D, Prati F, Cunto M, Iacono E, Merlo B. Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration. Theriogenology 2008;69:485–90. [15] Pukazhenthi B, Noiles E, Pelican K, Donoghue A, Wildt D, Howard J. Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors. Cryobiology 2000;40:139–50. [16] Tsutsui T, Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Murai M, et al. Unilateral intrauterine horn insemination of fresh semen in cats. J Vet Med Sci 2000;62:1241–5. [17] Howard JG, Barone MA, Donoghue AM, Wildt DE. The effect of preovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cat. J Reprod Fertil 1992;96:175–86. [18] Zambelli D, Cunto M. Transcervical artificial insemination in the cat. Theriogenology 2005;64:698–705.
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Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity _ n ski*, Ma1gorzata Ochota, Sylwia Prochowska, Wojciech Niza Agnieszka Partyka Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2014 Received in revised form 19 August 2014 Accepted 19 August 2014
This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 106/mL, 5 106/mL, and 1 106/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 106/mL dilution rate, spermatozoa diluted to 1 106 sperm/mL had a significantly lower proportion of motile (31.1% 19.8 and 0.7% 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% 3.1 and 69.1% 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% 11.9 vs. 75.9% 10.6) and chromatin status (defragmentation index, 3.3% 0.97 vs. 3.4% 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cat semen Sperm concentration Dilution effect Flow cytometry
1. Introduction The handling of semen and its processing associated with assisted reproductive technologies (ART) very often includes extension of sperm, as in the case of preparing spermatozoa for cryopreservation, IVF, flow cytometric assessment, or cell sorting by chromosomal sex. The latter two require high dilution, which may be detrimental for the motility and viability of spermatozoa, as is the case in bovine [1,2] and ram semen [3]. This phenomenon has been described as the “dilution effect” [4]. It is thought that the dilution effect is connected with a partial or even complete removal of some essential seminal
* Corresponding author. Tel.: þ48 71 3205315; fax: þ48 71 3205306. _ n ski). E-mail address: [email protected] (W. Niza 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.08.012
plasma (SP) compounds that protect the sperm from damage, thereby contributing to the deleterious effects of semen dilution. Although the role of SP is ambiguous and many authors reported both its beneficial and detrimental influence on sperm characteristics [5–7], the presence of SP seems to play a crucial role in the case of excessive semen dilution. The evidence in support of this hypothesis is that the re-addition of SP or its compounds to the highly diluted sperm can prevent or reduce its damage in sheep [3,8,9], rabbits [5], cattle [2,9], and pigs [9–12]. Articles dedicated to feline sperm dilution are scarce. Howard et al. [13] showed that even a twofold dilution of electroejaculated feline semen resulted in a decrease of sperm motility. To the authors’ knowledge, no other information on this topic is available, although the semen in this species is characterized by a high concentration of sperm cells and an extremely low volume of ejaculate
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ranging from 10 mL [14] to 200 mL [15]. Therefore, high dilution rates of semen are usually necessary before sperm cell assessment, especially in semen collected by urethral catheterization [14]. The dilution of semen is also necessary before artificial inseminationdinsemination dose for intrauterine insemination of queens varies from 4.2 106 to 10 106 of sperm cells deposited in 30 mL [16] up to 200 mL [17,18]. Flow cytometry, which is widely used in andrology laboratories, is a valuable tool for the assessment of semen [19]. One of the advantages of flow cytometry is the possibility to assess a large number (>10,000) of spermatozoa in a short period of time. However, it requires a relatively large sample volume. This problem is solved by sample dilution. In cats, the assessment of semen by flow cytometry is rarely used most likely because of a low ejaculate volume and because a small overall number of spermatozoa can be obtained. Depending on the collection method, the total sperm count varies from about 20 106 in the case of electroejaculation [20] and urethral catheterization [14] to 80.1 106 in the case of an artificial vagina [21]. Such a small number of spermatozoa per ejaculate is a limiting factor with reference to the parallel assessment of many sperm characteristics or to combine flow cytometry sperm evaluation with other procedures (e.g., cryopreservation or artificial insemination). The possibility of assessing highly diluted samples (1 106 cells/mL, as recommended by manufacturers of flow cytometers and fluorescent dyes) through flow cytometry may enable a simultaneous broad sperm evaluation and its use for ART. It could also widen the spectrum of the assessed sperm parameters. However, due to the fact that extreme dilution may influence semen characteristics, there is a theoretical risk of an artifactual change of the results of the semen evaluation. Consequently, the laboratory assessment of the fertilizing potential of a particular male may be erroneous. Urethral catheterization is a novel method of semen collection in cats, easy to introduce in everyday practice. In comparison with ejaculated spermatozoa (obtained via an artificial vagina or electroejaculation), semen collected using this method is characterized by a lower volume, higher sperm concentration, and a minimal presence of SP [14]. Thus, it very often requires a high dilution, but it has not been checked if such extensions may affect the sperm characteristics. Therefore, the aim of this study was to assess the influence of high sample dilution on spermatozoal motility, viability, acrosome integrity, and chromatin status in the feline urethral semen. 2. Materials and methods 2.1. Animals Spermatozoa were collected from eight privately owned male cats scheduled for routine castration procedures at the Department of Reproduction and Farm Animal Clinic of the Wroclaw University of Environmental and Life Sciences (Poland). All tomcats were clinically healthy Domestic Shorthair cats, aged between 8 and 36 months. All procedures were performed with the consent of the Second Local Ethical Committee in Wroclaw.
2.2. Semen collection Tomcats presented for routine orchidectomy were anesthetized using medetomidine hydrochloride im 80 mg/ kg of body weight (Sedator 1.0 mg/mL, Novartis, Poland) combined with ketamine im 5 mg/kg of body weight (VetKetam 100 mg/mL, VetAgro, Poland). To reduce the postoperative pain, an injection of meloxicam sc 0.3 mg/kg of body weight (Metacam 5 mg/mL, Boehringer Ingelheim Vetmedica, Germany) was given before the anesthesia. After the orchidectomy, the cats were given a mixture of benzathine benzylpenicillin 100,000 IU/mL, procaine benzylpenicillin 100,000 IU/mL, and dihydrostreptomycin sulfate 200 mg/mL im 1 mL/10 kg of body weight (Shotapen L.A., Virbac, France). The urethral semen was collected as previously described by Zambelli et al. [14]. Briefly, a tomcat urinary catheter with its tip cut to get a shorter, open-ended catheter was inserted approximately 9 cm into the urethra, taking care not to reach the bladder. Subsequently, the catheter was removed from the urethra for the purpose of collecting a semen sample. Immediately after successful sample collection, it was placed in a prewarmed Eppendorf tube containing 200 mL of semen extender based on TRIS buffer ((3.02%, wt/vol) TRIS (Sigma–Aldrich, Poland), 1.35% (wt/vol) citric acid (Sigma–Aldrich), 1.25% (wt/vol) fructose (Sigma–Aldrich), in bi-distilled water; pH 6.5). 2.3. Experimental design and semen evaluation Each sperm sample was assessed for sperm motility and sperm concentration immediately after collection. The sample was then divided into three aliquots and a semen extender was added to reach the final concentration of spermatozoa 10 106/mL, 5 106/mL, and 1 106/mL. The 1 106/mL dilution rate was chosen as recommended by manufacturers of flow cytometers and fluorescent dyes. The 10 106/mL was the highest possible concentration to prepare, according to the amount of sperm obtainable from a cat that allows the creation of three dilutions for comparison. The concentration 5 106/mL was the value in between. Immediately after dilution, each sample was assessed for sperm motility and then prepared for a flow cytometric evaluation of viability, acrosome integrity, and chromatin status. 2.3.1. Sperm motility and concentration In order to assess the motility, 10 mL of a sperm sample (raw semen and a sample from each of the investigated dilutions) was placed on a prewarmed slide and the percentage of motile sperm was subjectively estimated under a contrastphase microscope at a 200 magnification by three independent researchers and the mean value was calculated. To evaluate sperm concentration, a 10-mL aliquot of the semen sample was diluted in 200 mL of distilled water and cells were counted in 80 squares of the Thoma chamber. Counting was repeated in a second chamber and the mean value was calculated. 2.3.2. Flow cytometry assessment Measurements were carried out on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. The fluorescent probes were excited by an Argon ion 488 nm laser. Detection
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of green fluorescence was set with an FL1 band-pass filter (530 nm ⁄ 30 nm) and red fluorescence was measured using an FL2 longpass filter (>670 nm). Green florescence of acridine orange (double-stranded DNA) was detected on the FL1 detector and red fluorescence of acridine orange (AO; singlestranded DNA) was identified on the FL3 detector. Acquisitions were measured using the CellQuest 3.3 software (Becton Dickinson). The non-sperm events were gated out based on scatter properties and were not analyzed. A total of 20,000 events were analyzed for each sample. 2.3.2.1. Viability. The viability was assessed using the dual fluorescence staining with SYBR-14 and propidium iodide (PI) as described by Pukazhenthi et al. [15], with minor changes. Spermatozoa were stained using the sperm viability kit (Live/Dead Sperm Viability Kit, Life Technologies Ltd, Carlsbad, CA, USA). An aliquot of 300 mL of diluted samples was pipetted into cytometric tubes, and 2.5 mL of SYBR-14 working solution was added. The working solution was obtained by diluting a commercial solution of SYBR-14 in distilled water with a ratio of 1:49. Samples were mixed and incubated at room temperature in the dark for 10 minutes. Then, the cells were counterstained with 1.5 mL of PI 3 minutes before analysis. Four subpopulations of cells were observed on dot plots of PI/SYBR-14 fluorescence: dead spermatozoa (PIþ/SYBR14), moribund spermatozoa (PIþ/SYBR-14þ), live spermatozoa (PI/SYBR-14þ), and a nonlabeled population (PI/SYBR-14) that was considered to be debris [22]. 2.3.2.2. Acrosome integrity. The acrosome damage was assessed using lectin PNA from Arachis hypogaea Alexa Fluor 488 Conjugate (Life Technologies Ltd.). Because a protocol for cats has not yet been described, we used a modified protocol used in our laboratory for fowl semen [22]. Briefly, 5 mL of PNA working solution (1 mg/mL) was added to 500 mL of semen samples and incubated for 5 minutes at room temperature in the dark. Following incubation, the supernatant was removed by centrifugation (500 g for 3 minutes), and the sperm pellets were resuspended in 500 mL of TRIS buffer. For counterstaining, 1.5 mL of PI was added to samples before cytometric analysis. Dot plots of PNA/PI-stained spermatozoa showed four populations of cells: live cells with an intact acrosome (PI/ PNA), live cells with a ruptured acrosome (PI/PNAþ), dead cells with an intact acrosome (PIþ/PNA), and dead cells with a ruptured acrosome (PIþ/PNAþ) [22]. 2.3.2.3. Chromatin status. The AO stain was used to assess sperm DNA integrity as previously described in cats [23], with minor changes. The suspension (100 mL) was subjected to brief acid denaturation by mixing with 200 mL of a lysis solution (Triton X-100 0.1% (vol/vol), NaCl 0.15 M, HCl 0.08 M, pH 1.4), held for 30 seconds, and mixed with 600 mL of an AO solution (Life Technologies Ltd) (6 mg AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6). After 3 minutes, the samples were aspirated into a flow cytometer. The main population that represents spermatozoa with a predominantly normal double-stranded configuration of DNA was observed on dot plots. Sperm cells located to the
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right of this main population represent cells, which show an increased amount of red fluorescence, indicating denatured DNA (DFI). Spermatozoa above the upper border of the main cluster of the sperm population represent spermatozoa that have an abnormally high DNA stainability [22]. 2.4. Data analysis The results obtained are presented as mean SD of measurements. Data were analyzed using ANOVA and Duncan’s multiple range test. The level of significance was set at P value less than 0.05. An elaboration of tests was carried out using the Statistica software for Windows, StatSoft Polska Sp. z o.o. 3. Results 3.1. Sperm motility The motility of the raw semen was 66.9% 20.2. Sperm motility was reduced by increasing sperm dilution. Samples diluted to the concentration of 10 106/mL showed a motility decrease by at least 50% (average motility, 31.1% 19.8), which was further exacerbated in a concentration of 5 106/mL (average motility, 16.7% 18.5). In the most diluted samples (1 106/mL), single motile spermatozoa were observed (average motility 0.7% 1.6) or none at all. The results are presented in Figure 1. In many samples with a concentration level of 10 106/ mL and 5 106/mL, the spermatozoa showed no progressive movement. Only a subtle oscillation was observed, which could indicate an adherence of the motile spermatozoa to the glass surface. The magnitude of this phenomenon differed between samples. It was seen less frequently in specimens of high motility than those characterized by low motility, where this oscillation was often the only type of movement observed. 3.2. Flow cytometry assessment The results of viability, acrosome integrity, and chromatin status are shown in Table 1. The percentage of live cells (SYBRþ/PI) showed a statistically significant difference (P ¼ 0.016) between the 5 106/mL and 1 106/mL dilution rates and highly significant differences (P ¼ 0.009) between the 10 106/mL and 1 106/mL dilution rates. Simultaneously, the percentage of dead cells (SYBR/PIþ) differed significantly (P ¼ 0.02) between the 10 106/mL and 1 106/mL (6.5 3.2 vs. 21.3 11.9) dilution rates. There were no significant differences in terms of the acrosome integrity and chromatin status between different dilution rates in the assessed populations of spermatozoa. 4. Discussion The quality of cat urethral semen in our study was comparable to the results obtained by other authors in terms of motility (66.9% 20.1 in comparison with 78.1% 9.6 [14] and 50% 12.4 [20] and 50.4% 20.3 [24]) and viability (88.3% 3.1 compared with 82.2% 18.4 [24]
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Fig. 1. Motility of spermatozoa in different concentration levels.
a.b.c
Different superscripts within bars indicate significant differences, P < 0.05 (N ¼ 8).
and 80.0% 10.1 [14]). In our study, the results of the acrosome integrity were higher than those obtained by Filliers et al. [24], who used a fluorescent microscope (>90% vs. 84.2% 6.8, respectively). This difference may be caused by the type of the extender used and the differences in the anesthetic procedures, sperm preparation protocols, and evaluation methods [20]. This was the first study analyzing cat urethral semen in terms of chromatin integrity. Our findings obtained with the use of flow cytometry (normal DNA stainability, 93.4% 2.9) are similar to the results obtained by other authors with the use of AO staining and a fluorescent microscope for epididymal (95.2% 2.6) [25] and ejaculated (97.1% 2.1) [26] sperm cells. In the present study, we confirmed a deleterious effect of increasing dilution on sperm motility and viability that was previously described by many authors with reference to ejaculated bull [1,2,8], boar [10–12], and ram semen [3,8]. However, the underlying mechanism of this phenomenon has not yet been fully explained. In general, it is thought that the dilution of semen presumably removes adsorbed (“coating”) proteins, natural antioxidants, and other beneficial components in SP required for the maintenance of the membrane integrity and function of spermatozoa [27]. However, it seems that the mechanisms and factors responsible for the “dilution effect” are different when it comes to the loss of motility and loss of viability. In our study, the loss of motility induced by dilution was much more marked than the loss of viabilitydthe percentage of viable cells in the highest dilution was on average 69%, whereas the number of motile spermatozoa in this group decreased to 0.7%. Many other authors noticed that the viability and motility do not always correlatedsometimes viable spermatozoa were assessed as immotile and motile
ones were classified as nonviable [8,9,12]. Therefore, these two parameters should be interpreted separately. Factors like temperature, pH, and osmolality, either below or above physiological ranges, can impair spermatozoal motility [4,15], but as the negative effect of these factors is widely known, they will not be discussed here. Apart from these factors, proteins are thought to be essential for maintaining sperm motility. An evidence to support this hypothesis is that the deleterious effect of salt diluents on spermatozoa can be at least partly overcome by the inclusion of certain organic substances, mainly of a colloidal nature [4]. Moreover, the loss of motility in SPsubtracted samples can be restored by adding BSA [28,29]. In addition, the phenomenon of spermatozoa sticking to the glass surface, which was observed in our study as well as by other authors [6,29], may indicate that the dilution alters the sperm membrane surface proteins. Removal of these proteins creates charge differences, which promote agglutination [9]. Another explanation for the loss of motility in the diluted samples is a low level of motilitystimulating factors [29]. However, because semen extenders are nowadays supplemented with energy sources (glucose and fructose), citric acid, and many other constituents, the lack of these factors may be limited to cases where only simple diluents (e.g., normal saline) are used. Complex extenders can maintain spermatozoal motility equally well or even better than SP [30]. The matter is more intricate in the case of sperm viability. Ashworth et al. [3] reported that the survival of ram sperm in high dilution was improved by the addition of low molecular weight components such as sucrose, lactate, pyruvate, ethylene glycol tetraacetic acid (EGTA), Ca2þ, Mg2þ, and phosphate. Similarly, in the study of Catt et al. [8], a complex
Table 1 Plasma membrane integrity, acrosome integrity, and chromatin status of feline urethral spermatozoa in three dilution rates. Spermatozoa population
Live (SYBRþ/PI) Live with an intact acrosome (PI/PNA) Defragmentation index (%DFI) a,b
Concentration 10 106/mL
5 106/mL
1 106/mL
88.3 3.1a 76.7 11.9 3.3 0.97
85.9 4.0a 79.8 11.0 2.8 1.2
69.1 12.8b 75.9 10.6 3.4 1.7
Different superscripts within rows indicate significant differences, P < 0.05 (N ¼ 8).
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extenderdBeltsville thawing solutiondmaintained the viability of boar and ram spermatozoa, with or without the addition of SP. This suggests that simple metabolites or ionic components may be the most important components of SP for the maintenance of cell viability. Thus, the dilution effect on spermatozoa may be minimized by the use of wellbalanced semen extenders. However, seminal proteins are also important in the case of high semen dilution, as they are thought to exert the most significant response to SP [31]. The content and function of seminal proteins have been intensively studied for last 20 years. As a result, many proteins were identified and described (for review see [32] and [33]). A positive or negative correlation of particular proteins with male fertility [34,35], sperm quality [36–38], and freezability [39,40] has been reported. However, the role of proteins in maintaining spermatozoal viability in high dilution has not, to date, been fully explained. In general, they are considered to stabilize sperm cells by coating the sperm surface [41,42]. These “coating factors” tend to be eluted from spermatozoa on dilution, resulting in destabilization of their plasma membranes leading to their subsequent death [3]. Contrary to motility, the effect is highly specific, linked not with the presence of proteins in general, but with a defined protein. This was proved in the case of ram semen, where the addition of bovine serum albumin (65–70 kDa) did not prevent the loss of viability, instead beneficial agents that protected spermatozoa from the dilution effect were found in 5 to 10 kDa fraction of SP [3]. This subject was studied deeper in boars and it was shown that the addition of an isolated nonheparin–binding spermadhesin PSP-I/PSP-II heterodimer to extremely diluted semen resulted in a greater percentage of viable and motile cells, whereas the addition of heparinbinding spermadhesin (HBP) had a substantial detrimental effect on the survival of spermatozoa [12]. Studies dedicated to SP proteins in cats are limited [20], and, to the authors’ knowledge, no information about its protective or detrimental influence on highly diluted semen is available. Our results suggest that although urethral semen is characterized by a minimal presence of SP [14], probably some plasma compounds are present in urethral semen and play an important role in spermatozoal protection against high dilution. Further studies on sperm and SP proteomics are required to establish the exact agents. Proteins are responsible not only for maintaining sperm motility and membrane integrity but also for sperm capacitation and fertilization of the oocyte (for the review see [43]). It is well known that SP possesses factors that can decapacitate sperm cells and prevent/inhibit the acrosome reaction (reviewed by [44]). This effect is undesirable in case of in-vitro fertilization; therefore, removal of SP is a routine procedure before IVF [45]. On the other hand, both cooling and cryopreservation cause capacitation-likechanges, which may interfere with fertilizing potential of sperm cells. These changes can be reversed or minimized by the addition of SP [46–48]. In our study, higher dilution rates did not cause changes in the acrosome integrity, which is consistent with results obtained by Catt et al. [8] when analyzing fresh ram and boar semen and contrary to the results for cryopreserved bull spermatozoa [49]. The supplementation of SP to ram semen lowered the number
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of acrosome-reacted cells after sex sorting [9], but not after cryopreservation of sex-sorted sperm [50]. These discrepancies in the influence of SP on acrosome status may be a result of different experimental conditions, such as the level of dilution/semen concentration, additional processing of semen (sex –sorting and cryopreservation), methods of evaluating acrosome integrity, species-specific characteristics, and other factors that are discussed below. In our study, dilution did not affect the chromatin structure, which is in agreement with the results for SP removal in dogs [51] and stallions [52]. However, in stallion removal of SP was protective to sperm DNA integrity during 24 and 48 hours cooled-storage [52]. In dogs converselydremoval of SP caused significant increase in %DFI after 3 hours of post-thaw incubation [51]. In humans, a fivefold dilution decreased sperm nuclear resistance to decondensation in SDS [53]. These examples show that semen dilution or SP removal can have an influence on sperm DNA, but the effect depends on other conditions like time and species factor, which are discussed below. It must be emphasized that our study refers only to the high rates of sperm dilution. Moderate dilution of electroejaculated cat semen had no effect on sperm parameters [54,55]. Similar results were obtained by Castellini et al. [5] in rabbits – two- to fivefold SP dilution increased motility parameters, but further dilution caused sperm deterioration. Other studies revealed that the optimal level of SP contents is 10%d a higher concentrations of SP did not further enhance spermatozoal survival [3], had no effect on the viability [6], or even appeared to be detrimental to the sperm motility and membrane integrity [9]. This indicates that SP contains not only beneficial agents but also detrimental ones (like some specific proteins [12], leukocytesda source of ROS [56,57], bacteria [58], hemoglobin [59]), and an excessive amount of SP may have a negative effect to sperm cells. Similarly, it must be taken into consideration that our study examined only a short-time effect of dilutiondup to 30 minutes (the time of the semen assessment). The immediate detrimental effect, which we observed, was also described by other researchers [3,5,11]. This effect is an important factor when assessing fresh sperm. In this case, the role of SP may be considered positive. However, ART requires very often prolonged storage of semen. In those conditions, the influence of SP and its dilution may be different. In vivo spermatozoa have only short-lasting contact with SP, as they quickly swim free to further parts of the female tract. Therefore, a long contact of spermatozoa with SP, as in the case of ART, seems to be unnatural. During the storage period, spermatozoa are exposed to potential detrimental components included in SP for a long time and, simultaneously, protective agents may be “used up.” Hence, the effect of SP dilution/removal/addition during long incubation and cold storage has already been the point of interest of many researches, with various results [28,49,60–64]. In general, the presence of SP was detrimental to sperm cells [61,62,64], although partial removal gave better results than either leaving whole SP or its complete removal [60,62], which is in agreement with the previous paragraph. Another aspect that has to be considered is that fresh semen was assessed in our study. Numerous studies have
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reported a deleterious influence of SP on semen cryopreservation results [7,65–67], particularly in males characterized by poor freezability [68]. Hence, SP is usually removed before freezing [69]. Studies involving post-thaw semen dilution have been contradictory. For example, in rams, post-thaw dilution decreased the viability and motility of spermatozoa [66]. When SP was removed before freezing, post-thaw dilution resulted in better semen quality in cats [70] and dogs [71]. In cats, resuspension of cryopreserved epididymal spermatozoa in SP gave worse results than resuspension in TRIS [72]. On the one hand, semen extension after freezing– thawing can dilute detrimental factors like ROS, hemoglobin, and metabolites accumulated during the freezing procedure and is consequently considered to be beneficial. On the other hand, it was shown that SP can restore the membrane damaged during cryopreservation [73]. In addition, the type of semen (e.g., ejaculated and epididymal) and semen collection method (whole ejaculate and only sperm-rich fraction) can affect the results, as semen obtained using different methods varies in SP content and concentration [20]. Although there are numerous studies comparing different types of semen, none of them (to the authors’ knowledge) refers directly to the high semen dilution. When studying SP addition, results were contradictory. For example, SP addition to epididymal sperm before freezing–thawing had positive effects in the Iberian red deer [74] but did not improve sperm quality in a cat [75]. In the stallion, SP had a stimulatory effect on the motility of epididymal spermatozoa during cooled storage but depressed motility in ejaculated spermatozoa [76]. In this study, we used feline urethral semen. Our observations for epididymal sperm suggest that dilution in a TRIS-buffered semen extender had none or much lesser influence on this kind of semen (Prochowska, unpublished results). We discussed our results for feline semen in reference to other species (due to the lack of articles dedicated to the cat), but one should bear in mind that there are species-specific differences in the susceptibility of spermatozoa to damagedram sperm cells are more sensitive to dilution than boar and bull ones [3,8]. The differences in the composition of SP from different species [77] could explain this and other species-specific differences in terms of the benefits of SP [6,9] and the negative effect of adding heterologous SP [8]. Moreover, semen composition differs not only among species but also among and within individuals [9,35,37,39]. The variability in SP contents can be influenced by the season [36] and the methods of semen collection [20]. From this point of view, the absence, presence, under- or overexpression, and critical concentration of specific proteins in the ejaculate of individual males could alter sperm functions. This may explain the variability of results obtained by different authors for the same species (e.g., [5] and [6] for rabbits; [1] and [49] for bulls), for different males [2,9,10,49] and different ejaculates of the same male [1]. 4.1. Conclusion Feline urethral semen is susceptible to the dilution shockdin highly diluted samples the number of motile and membrane intact cells decreased. This immediate change of viability and motility in highly extended samples suggests
that these features can be easily artifactually changed. Such possibility should be taken into consideration during semen assessment and when comparing results obtained by different authors. Acknowledgments We acknowledge the financial support from MNiSW/ NCN N N308 576540 (N308 576540). The authors are grateful to Iwona Zbyryt (MSc) for her help with the flow cytometric analysis and to Barbara Smalec (MSc) for her excellent technical assistance. References [1] Garner DL, Thomas CA, Allen CH. Effect of semen dilution on bovine sperm viability as determined by dual-DNA staining and flow cytometry. J Androl 1997;18:324–31. [2] Garner DL, Thomas CA, Gravance CG, Marshall CE, DeJarnette JM, Allen CH. Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology 2001;56:31–40. [3] Ashworth PJ, Harrison RA, Miller NG, Plummer JM, Watson PF. Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod Fertil Dev 1994;6:173–80. [4] Mann T. The biochemistry of semen and of the male reproductive tract. London: Methuen and Co.; 1964. [5] Castellini C, Lattaioli P, Moroni M, Minelli A. Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa. Anim Reprod Sci 2000;63:275–82. [6] Dott HM, Harrison RAP, Foster GCA. The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma. J Reprod Fertil 1979;55:113–24. [7] Moore AI, Squires EL, Graham JK. Effect of seminal plasma on the cryopreservation of equine spermatozoa. Theriogenology 2005;63: 2372–81. [8] Catt SL, O’Brien JK, Maxwell WMC, Evans G. Assessment of ram and boar spermatozoa during cell-sorting by flow cytometry. Reprod Domest Anim 1997;32:251–8. [9] Maxwell WM, Welch GR, Johnson LA. Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Dev 1997;8:1165–78. [10] Caballero I, Vázquez JM, Centurión F, Rodríguez-Martínez H, Parrilla I, Cuello C, et al. Comparative effects of autologous and homologous seminal plasma on the viability of largely extended boar spermatozoa. Reprod Domest Anim 2004;39:370–5. [11] Caballero I, Vázquez JM, García EM, Roca J, Martínez EA, Calvete JJ, et al. Immunolocalization and possible functional role of PSP-I PSP-II heterodimer in highly extended boar spermatozoa. J Androl 2006; 27:766–73. [12] Centurion F, Vazquez JM, Calvete JJ, Roca J, Sanz L, Parrilla I, et al. Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution. Biol Reprod 2003;69:640–6. [13] Howard JG, Brown JL, Bush M, Wildt DE. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoa motility and morphology after swim up processing. J Androl 1990;11:204–15. [14] Zambelli D, Prati F, Cunto M, Iacono E, Merlo B. Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration. Theriogenology 2008;69:485–90. [15] Pukazhenthi B, Noiles E, Pelican K, Donoghue A, Wildt D, Howard J. Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors. Cryobiology 2000;40:139–50. [16] Tsutsui T, Tanaka A, Takagi Y, Nakagawa K, Fujimoto Y, Murai M, et al. Unilateral intrauterine horn insemination of fresh semen in cats. J Vet Med Sci 2000;62:1241–5. [17] Howard JG, Barone MA, Donoghue AM, Wildt DE. The effect of preovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cat. J Reprod Fertil 1992;96:175–86. [18] Zambelli D, Cunto M. Transcervical artificial insemination in the cat. Theriogenology 2005;64:698–705.
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