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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

The hypo-osmotic swelling test (HOS test) is a simple and inexpensive test to evaluate the functional integrity of sperm cell membranes. According to the existing literature, its simple applicability has turned it into a valuable additional parameter to standard canine semen analysis. In the recent years, much research has been conducted in this field. The aim of this systematic review was to evaluate the quality of published literature in canine reproduction concerning the HOS test. Using two distinguished databases, 38 articles were detected and analysed subsequently according to various aspects, for example study design, population, semen sampling and implementation concerning the HOS test. Although there are numerous articles available, the diagnostic value of the HOS test remains ambiguous. Until now, neither a recognized test protocol nor reliable reference values have been defined. Most of the trials evaluated show serious methodological flaws and therefore do not permit drawing reliable conclusions. According to our results, approximately half of the studies (n = 20) included a sample size of five or less animals. None of the studies examined the inter- or intraobserver agreement for the HOS test. Further research is warranted including appropriate statistical methods and a sufficient number of animals to establish a standardized test protocol as well as reliable reference values. Most importantly, it is required to clarify a correlation between the HOS test and the fertilizing capacity to determine the diagnostic value of the HOS test.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

The hypo-osmotic swelling test (HOS test) is a simple, inexpensive and easily applicable test to assess the functional integrity of the plasma membrane of sperm cells (Kumi-Diaka 1993; Quintela et al. 2010). Moreover, the HOS test is considered to be reliable and sensitive (Jeyendran et al. 1984) and to represent a valuable tool for routine semen evaluation (Rodriguez-Gil et al. 1994; Goericke-Pesch and Failing 2013).

The HOS test is based on the semi-permeability of intact cell membranes. According to Petrunkina et al. (2001), functional membranes are closely related to sperm function, as only intact sperm cells are able to adjust in the female tract. The spermatozoon is exposed to major osmotic differences at various stages in its life cycle. During ejaculation, sperm cells are transported from the hyperosmotic environment of the cauda epididymis with an osmotic pressure of 420 mmol/kg (Yeung et al. 1999) to the almost iso-osmotic fluids of seminal plasma and female genital tract with an osmotic pressure of 330 mmol/kg (Yeung et al. 2000). To withstand these osmotic challenges, intact membranes of sperm cells are able to regulate volume changes by the adjustment of intracellular ion concentration (Jeyendran et al. 1984; Petrunkina et al. 2004a). The functional integrity of sperm cell membranes can be determined by the HOS test (Jeyendran et al. 1984).

When performing the HOS test, spermatozoa are exposed to hypo-osmotic solutions. Under these conditions, spermatozoa swell due to influx of water until equilibrium is reached, resulting in the curling of sperm tails (Rodriguez-Gil et al. 1994). After incubation, replicate slides are examined under a phase-contrast microscope. Afterwards, the number of non-swollen (i.e. damaged membranes) and swollen (i.e. intact membranes) cells can be counted (World Health Organization 2010).

In the recent years, many studies have been conducted utilizing the HOS test to evaluate the quality of canine spermatozoa. However, a recognized test protocol or reference values are not available. Therefore, the overall objective of this study was to systematically review studies using the HOS test in dogs. Specifically, we set out to evaluate the available literature according to certain quality parameters and test characteristics, such as randomization, blinding, inter- and intraobserver agreement and sample size.

Material and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

A comprehensive literature search was conducted on 28th March 2012 utilizing the databases CAB (http://ovidsp.tx.ovid.com) and PubMed (http://www.ncbi.nlm.nih.gov/pubmed) to identify the literature related to the analysis of canine semen. The subject heading ‘dogs + semen analysis’ was applied. No limits were set. All references were imported into EndNote (version X4.0.2; Thomson Reuters EndNote, New York, NY, USA).

Using an automatic function provided by EndNote, duplicates and reviews were identified and excluded. Duplicates and reviews that were not recognized by EndNote were deleted manually. Only studies performing the HOS test on canine semen were included. Additional records were identified through references in the included articles to cover the pertaining literature as wide as possible. All selected articles were systematically reviewed considering preset criteria. Relevant aspects concerning the study design, such as sample size, number of ejaculates, blinding and randomization were assessed according to Sannmann et al. (2012). Furthermore, data concerning the population of the dogs, that is, selection criteria, housing, feeding, age, breed and health status were extracted. Additionally, information regarding semen analysis, such as the applied sperm fraction and whether pooled semen were employed, were collected.

Furthermore, a systematic follow-up of citations concerning the HOS test in the eligible articles was conducted to generate a citation map. Every quotation regarding the HOS test was entered into a spreadsheet (Microsoft Excel 2010; Microsoft Deutschland GmbH, Munich, Germany) irrespective of species. Only publications quoted more than three times and related to canine reproduction as well as those which were essential to determine the origin of quotation of the HOS test were included in the citation map. For the development and presentation, an open source mind-mapping software (XMind 2012; XMind Ltd, Hong Kong, China) was used.

To gain an overview of different methodologies for HOS testing, osmolarities, times of incubation, number of examined sperm cells and magnification were documented. Further parameters assigned for semen evaluation, such as motility, concentration, viability and morphology were also considered. All extracted data were entered into a spreadsheet (Microsoft Excel 2010; Microsoft Deutschland GmbH).

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Literature search

Using the subject heading ‘dogs + semen analysis’, a total of 354 references were found (CAB: 29, PubMed: 325). After eliminating duplicates (n = 12) and reviews (n = 23), 319 articles remained. Articles regarding other species than dogs (n = 9) and articles not focusing on semen analysis (n = 35) were excluded as well. One article could not be examined as it was written in Korean language. After excluding trials not performing the HOS test (n = 247), 27 articles remained. Additionally, 11 articles were identified through a systematic review of citations in the retrieved papers, leading to a total of 38 articles being eligible for final analysis. A systematic follow-up of citations concerning the HOS test in the eligible articles was conducted to identify the origin of its quotation (Fig. 1).

image

Figure 1. Citation map of studies investigating the origin of the documentation of the HOS test in dogs. Boxes with dashed lines: papers not being included in the study because of different species. Boxes with solid lines: papers investigated

Download figure to PowerPoint

Of the 38 papers evaluated, 27 were published in the last decade. The articles were published in 13 different journals focusing on animal reproduction, such as Theriogenology (n = 18) and Animal Reproduction Science (n = 5). The majority of these journals (11/13) were peer reviewed; thus, 94.7% of the articles were published in peer-reviewed journals. Seven journals (53.9%) have an impact factor above 1.0.

Study design

All articles were based on prospective studies. None of the 38 included studies was specified as being randomized, and only one study was identified as blinded (Quintela et al. 2010). Reference values for results of the HOS test were defined in one study (Riesenbeck et al. 2001). None of the studies examined the inter- or intraobserver agreement for the HOS test. Only Rota (Rota et al. 2005, 2006) calculated correlations between two observers based on 15 (three ejaculates from five dogs; Rota et al. 2005) and 10 samples from five dogs, respectively (Rota et al. 2006). The correlations vary between r = 0.83, p < 0.001 (Rota et al. 2005) and r = 0.844, p < 0.001(Rota et al. 2006). In 21 studies, no information was provided on inclusion or exclusion criteria of the dogs involved.

Population

Of 38 studies, 20 (52.6%) included five or less dogs and seven (18.4%) included six to 10 dogs. Nine trials (23.7%) included more than 10 dogs whereas in two articles the population was not defined. The dogs were aged between 10 months and 8 years, if specified (n = 26). Predominately, beagles were used (12 of 31 studies with species information). For the duration of the studies, the dogs were kept either in indoor or outdoor kennels (n = 15), or lived together with their private owners (n = 3). In 20 articles, housing was not defined. Dogs were commonly fed with commercially balanced diets. In 26 articles, the ration was not specified. Semen was collected by digital manipulation (n = 30), by aspiration after castration (n = 3) or using an artificial vagina (n = 1). Sampling was accomplished at different frequencies, primarily once or twice weekly. In 16 papers, sampling frequency was not specified. In 11 trials, 20 or less ejaculates were included whereas in 16 studies the number of ejaculates was not specified. Only four articles (10.5%) evaluated more than 40 ejaculates. Pooling of semen samples was conducted in six studies. The majority (n = 27) of studies separated the three fractions of the ejaculates (i.e. pre-sperm fraction, sperm-rich fraction, prostatic fraction). In eight articles, the tested fraction was not specified. The second fraction was used for the HOS test in 24 studies. In three cases, semen samples were taken by aspiration after castration. Consequently, number and fraction of the ejaculates as well as precognition of these dogs could not be determined.

HOS-test protocols

To evaluate the functional integrity of human spermatozoa, Jeyendran et al. (1984) used ejaculates from three men divided into 35 aliquots and exposed these to different osmotic solutions. According to the authors, solutions at 150 mOsmol led to optimal and repeatable results.

To appraise functional integrity of canine spermatozoa, two authors introduced the HOS test to veterinary medicine (England and Plummer 1993; Kumi-Diaka 1993). Kumi-Diaka (1993) examined ejaculates of eight dogs. Canine spermatozoa were submitted to solutions of different osmolarities. According to their results, it was recommended to incubate solutions at 60 mOsmol and 37°C for 45 min.

For the method described by England and Plummer (1995), two ejaculates were collected from each of the six dogs. Referring to Jeyendran et al. (1984), canine spermatozoa were exposed to solutions with different osmolarities. Similar to the results postulated by Jeyendran et al. (1984), a solution of 150 mOsmol incubated at 37°C for 30 min generated a high proportion of swollen spermatozoa.

Both authors considered the HOS test to be a reasonable assay to assess membrane integrity. Protocols of various studies (20/35) conducted afterwards are referring to these reports (Table 1).

Table 1. Methodological parameters applied by the analysed articles concerning the HOS test in dogs
Author (year)OsmolaritiesIncubation
mOsmol/l°Cmin
Mota et al. (2011)03845
Bencharif et al. (2010)1003760
Kim et al. (2010a)1003745
Kim et al. (2010b)1003745
Futino et al. (2010)1503730
Quintela et al. (2010)603745
Lopes et al. (2009b)03845
Corral-Baques et al. (2009)150; 3003715
Michael et al. (2009)60Not given45
Lopes et al. (2009a)1503730
Michael et al. (2008)60Not given45
Pinto and Kozink (2008)100371;60
Michael et al. (2007)60Not given45
De Souza et al. (2007)1503730
Tittarelli et al. (2006)Not givenNot givenNot given
Oliveira et al. (2006)60Not givenNot given
Silva et al. (2006)1503845
Rota et al. (2006)603745
Rota et al. (2005)603745
Petrunkina et al. (2005)180; 300; 450395;20
Corral-Baques et al. (2005)1503715
Petrunkina et al. (2004a)180; 300; 450395;20
Nur et al. (2004)1003760
Uysal and Korkmaz (2004)60370;5;30;60
Hishinuma and Sekine (2004)038.55
Petrunkina et al. (2004b)150; 30025;33;395
Hishinuma and Sekine (2003)0; 15038.55; 60
Riesenbeck et al. (2001)1503730
Saratsis et al. (2000)603745
Mogas et al. (1998)1003710–120
Rota et al. (1995)603745
England (1995)Not givenNot givenNot given
Rodriguez-Gil et al. (1994)100; 150; 30037Not given
Kumi-Diaka and Badtram (1994)60Not given60
Kumi-Diaka and Harris (1994)60; 100; 150; 200; 300Not givenNot given
Kumi-Diaka (1993)60; 100; 150; 200; 30035;3715;30;45;60;75;90
England and Plummer (1993)0; 50; 100; 150; 200; 250; 3003730
von Kölliker (1856)0Not givenNot given

Additional parameters

In every trial investigated (n = 38), sperm quality was evaluated by combining HOS test with additional parameters. In 36, 34 and 30 cases, the HOS test was combined with motility, morphology and concentration, respectively. The majority of trials (n = 27) combined all four parameters. In 10 studies, correlations between HOS test and motility were calculated ranging from r = 0.35, p < 0.05 (Rota et al. 2005) to r = 0.98, p < 0.002 (Kumi-Diaka and Badtram 1994). Also, correlations existed between HOS test and viability in fresh semen [n = 2; r = 0.68, p < 0.05 (Pinto and Kozink 2008) and r = 0.6825, p ≤ 0.005 (Rodriguez-Gil et al. 1994)] or acrosome reaction in fresh semen [n = 2, r = 0.83, p < 0.05 (Kumi-Diaka and Badtram 1994) and r = 0.72, p < 0.03 (Petrunkina et al. 2004a)].

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

In the past, dog breeding has experienced increasing interest and cryopreservation of canine semen appears to be more important than ever (Kim et al. 2010b). Functional sperm cell membranes are crucial for successful freezing and thawing processes. With regard to the HOS test, many researches have been carried out especially in the last decade. It's diagnostic value, however, still remains ambiguous. Until now, neither a standardized test protocol nor reliable reference values for the HOS test have been defined.

When accomplishing a systematic review, it is important to consider the study designs of the articles (Khan et al. 2003). Study designs, that is, procedures under which studies are carried out, should be set-up in a way that bias is unlikely to occur. Consequently, studies concerning diagnostic tests should describe the methods of testing accurately to assure reproducibility (Watson and Petrie 2010). The majority of the evaluated articles provided detailed information on equipment and methodological procedures. However, there was only little information provided concerning selection criteria and background of the dogs enrolled.

To establish a new, reliable diagnostic method, it is important to assess the repeatability and reproducibility of the measurement process (Watson and Petrie 2010). Repeatability is defined as an agreement between two measurements on the same samples, whereas reproducibility means that two individuals are using the identical methodology on identical samples (Watson and Petrie 2010). Our findings reveal that except for Rota (Rota et al. 2005, 2006), neither an intra- nor an interobserver agreement for the HOS test was determined. According to our results, 20 (52.6%) of the studies included a sample size of only five or less animals. Merely nine trials (23.7%) included more than ten animals. More important than the absolute number of animals included is the question whether the sample size of each group was adequate relative to the scope of the study (Lenth 2001). In fact, eight studies used semen, which was pooled and therefore did not analyse the individual semen quality in each dog (Batista et al. 2012). We conclude that sample size as one important component of evidence was either not addressed at all or number of dogs was marginal in more than half of the studies performed. Overall, most of the studies investigated show serious methodological flaws and therefore do not permit drawing reliable conclusions.

When examining the implementation protocols for HOS testing, parameters, such as osmolarity, incubation time and incubation temperature varied from study to study. Based on their results, Kumi-Diaka (1993) and England (1995) recommended diverging parameters. Therefore, all following HOS studies either established their protocols on the basis of these two recommendations or generated new parameters. It is unclear, however, if the utilization of different parameters provide comparable results. To minimize potential bias, it is required to develop standardized test protocols (Watson and Petrie 2010).

To our knowledge, no reliable reference values were defined for HOS test in dogs. Only one attempt has been made (Riesenbeck et al. 2001) to determine a reference value, which has not been confirmed yet. Furthermore, it is still not possible to interpret the diagnostic value of the HOS test to predict the fertilizing capacity. Recently, one study compared two different freezing extenders (Bencharif et al. 2010) and investigated standard semen parameters, membrane function and fertility. All of the six bitches were inseminated successfully. A correlation between HOS test and fertilizing capacity, however, was not investigated. Currently, another study (Goericke-Pesch and Failing 2013) analysed retrospectively canine semen evaluations with emphasis on the use of the HOS test in 256 dogs (400 ejaculates). Significant correlations were found between HOS test and motility (p < 0.0001), viability (p < 0.0001), age (p < 0.001), acrosomal status (p < 0.5), pathomorphology (p < 0.0001) and sperm concentration (p = 0.011). A possible correlation between HOS test and fertilizing capacity was assumed by the authors. Our systematic literature assessment demonstrates that a correlation between HOS-test response and fertilizing capacity as reported in humans (Jeyendran et al. 1992) has not yet been established in dogs.

Several authors conclude that the HOS test represents an additional tool to conventional evaluation of semen quality despite a dearth of information regarding its diagnostic value. Most of the studies investigated have serious methodological flaws (e.g. limited sample size, no randomization, no inter- or intraobserver agreement) and therefore do not permit drawing reliable conclusions. Until now, neither a standardized test protocol nor reliable reference values have been defined. Further research is warranted including appropriate statistical methods and a sufficient number of animals to establish a standardized test protocol as well as reliable reference values. Most importantly, it is required to clarify a correlation between HOS test and fertilizing capacity to determine the diagnostic value of the HOS test.

Acknowledgement

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

The authors thank Camillo Krawczyk from the veterinary library of the Free University Berlin for his extraordinary capability at locating articles.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Sandra Karger: designed study, analysed data, drafted paper. Sebastian Arlt: designed study, analysed data, drafted paper. Peggy Haimerl: designed study, drafted paper. Wolfgang Heuwieser: designed study, drafted paper.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References
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