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Contents

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

The aim of this study was to evaluate the influence of Hoechst 33342 (H-42) concentration and of the male donor on the efficiency of sex-sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 106 sperm/ml, split into four aliquots, stained with increasing H-42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non-viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X- and Y-chromosome-bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H-42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H-42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex-sorting of canine spermatozoa by flow cytometry can be performed successfully using H-42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.


Introduction

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

The sexing of mammalian spermatozoa is an emerging reproductive technology based on the difference in DNA content between X- and Y-chromosome-bearing spermatozoa (Johnson et al. 1989). This difference enables the separation of spermatozoa using a flow cytometer/cell sorter and a DNA-specific fluorochrome, Hoechst 33342 (H-42), which allows measuring DNA content on the basis of variations in the fluorescence intensity emitted by X- and Y-chromosome-bearing spermatozoa (Keeler et al. 1983; Seidel and Garner 2002).

Sperm sex-sorting technology has been successfully used to produce offspring of a pre-determined sex in many domestic species, including cattle, swine, sheep and horses (Garner 2006). Recent studies have been performed with the aim of extending sperm sexing technology to other domestic species, such as cats (Spinaci et al. 2007; Pope et al. 2009), and to endangered and exotic mammalian species, such as primates (O′Brien et al. 2005), dolphins (O′Brien and Robeck 2006); alpacas (Morton et al. 2008), elephants (Hermes et al. 2009), buffalo (Lu et al. 2010) and rhinoceroses (Behr et al. 2009).

The development and study of sex-sorting technology in dogs could have numerous benefits and applications. Currently, artificial insemination (AI) and semen conservation are available to, and increasingly demanded by, dog breeders and owners worldwide, contributing to a growing interest in the marketing of canine semen (Thomassen and Farstad 2009). Therefore, the possible association of an emerging reproductive technology such as the sex-sorting of spermatozoa with other routine reproductive techniques, such as AI or cryopreservation, could optimize the profitability of the breeding and production of purebred dogs. The major motivation for the application of flow cytometry technology to the sorting of canine spermatozoa is the economic interest of breeders in generating pets of a desired sex. This technology could also be applied to the production of dogs used to assist humans with impaired senses or dogs trained in search and rescue because sex-related temperamental choices often underlie the selection of these animals (Garner and Seidel 2003; Meyers et al. 2008; Oi et al. 2013). The application of sex-sorting technology to the breeding of domestic dogs could be used as a model for the rescue of endangered canids, such as the African wild dog (Lycaon pictus) (Johnston et al. 2007) and wolf subspecies. At present, human intervention is crucial to the survival of these canids (Thomassen and Farstad 2009).

Despite these potential applications and the existence of published data on the birth of puppies bred using sexed semen (Meyers et al. 2008), information on canine spermatozoa sex-sorting is scarce. To the best of our knowledge, no data about canine-specific protocols for spermatozoa sex-sorting are available. Because the application of sexing technology to a new species involves the development of species-specific protocols (Maxwell et al. 2004), optimizing the H-42 staining concentration for the best resolution to properly identify X- and Y-chromosome-bearing populations of spermatozoa is a first step towards the application of this technology in dogs.

The aim of this study was to design an effective protocol for canine spermatozoa sex-sorting using flow cytometry. For this purpose, two specific objectives were set: (i) to optimize H-42 staining concentrations for the flow cytometric identification and separation of X- and Y-chromosome-bearing canine spermatozoa; and (ii) to determine whether variations between dogs exist that affect the sex-sorting of spermatozoa. Data on sorting parameters and the quality of spermatozoa after sex-sorting were assessed to determine the efficiency of the developed sorting procedure.

Materials and Methods

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

Reagents and media

All of the chemicals used in this study were analytical grade and, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All media were prepared under sterile conditions in a laminar flow hood (MicroH; Telstar, Terrasa, Spain).

The basic extender used to dilute canine spermatozoa was tris–citrate–fructose extender (TCF-Ext: Trizma base 200 mm; citric acid 63 mm; fructose 70 mm; pH 6.7; 324 ± 3 mOsm/kg), supplemented with 1000 IU/ml of penicillin and 1 mg/ml of streptomycin sulphate (Hemansson and Linde-Forsberg 2006). For the collection of sorted spermatozoa, TCF-Ext supplemented with 20% egg yolk (v/v) was used (TCF-EY Ext; Hemansson and Linde-Forsberg 2006).

Phosphate-buffered saline solution (PBS: 137 mm NaCl; 2.7 mm KCl; 0.86 mm KH2PO4; 6.4 mm Na2HPO4·7H2O; pH 6.8; 289 ± 4 mOsm/kg) was used to dilute the fluorochromes used for the flow cytometry evaluation of the quality of spermatozoa.

Animals and semen collection

All experimental protocols were performed in accordance with Directive 2000/63/EU EEC for animal experiments, and all experiments were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia (Spain).

A total of six males (Canis lupus familiaris) of various breeds (one Golden Retriever, one French Bulldog, one Ibizan Hound, one Weimaraner and two Scottish Terriers), ranging between two and 3 years of age and with proven fertility, were included in this study. The animals were clinically healthy and were routinely used for semen collection. The sperm-rich fractions (SRFs) of the ejaculates were collected into a pre-warmed calibrated plastic tube by digital manipulation, as described by Kutzler (2005). All of the ejaculates used in this study met the following quality criteria: at least 75% total motility, 60% progressive motility and 80% normal morphology.

Preparation of semen samples for sex-sorting

Immediately after semen collection, SRFs were diluted 1:2 v/v in TCF-Ext and were washed by centrifugation (700 g/5 min; Megafuge 1.0 R, Heraeus, Hanau, Germany) at room temperature (23°C). The supernatant was then removed, and the pellet was resuspended in TCF-Ext. Concentrations of spermatozoa were evaluated using a SP-100 NucleoCounter (ChemoMetec DJ, Allerod, Denmark), and the samples were rediluted with TCF-Ext to a concentration of 100 × 106 spermatozoa/ml. The extended semen samples were processed and stained with increasing concentrations of H-42, as described in the experimental design. After the addition of H-42, the spermatozoa were incubated in a water bath for 1 h at 36°C in the dark. Then, before being passed through the flow sorter, the stained spermatozoa were filtered through a 30-μm nylon mesh filter (Partec CellTrics, Gorlitz, Germany) to remove debris or clumped spermatozoa. Next, 1 μl of a 1 mg/ml food colouring dye (FD&C#40; Warner Jenkinson, St. Louis, MO, USA) was added to each sample to identify non-viable spermatozoa in the samples by quenching the H-42 fluorescence, and non-viable spermatozoa were excluded by dead cell gating during the sorting process (Morris et al. 2003; Clulow et al. 2012).

Flow cytometric sperm sex-sorting

A MoFlo SX® flow cytometer/sperm sorter (Dako-Cytomation Inc., Fort Collins, CO, USA) equipped with an ultraviolet wavelength laser (351–364 nm; Spectra Physics 1330, Mountain View, CA, USA) set to 175-mW output was used to identify and separate X-chromosome-bearing spermatozoa. Phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, 8.1 mm Na2HPO4, with 0.058 g/l penicillin G and 0.05 g/l streptomycin sulphate; pH 7–7.1, 285–310 mOsm), supplemented with 0.1% EDTA (Del Olmo et al. 2013), served as the sheath fluid, and the instrument sheath pressure was set to 40 psi. Gates were placed around correctly oriented spermatozoa to achieve >85% purity in the X-chromosome-bearing population of spermatozoa.

Analysis of sex-sorting characteristics was performed as previously described by Clulow et al. (2009). During sex-sorting, the software program used (SUMMIT; DakoCytomation, Fort Collins, CO, USA) produced a graphical representation of the spermatozoa as they were detected by the flow cytometer. Forward (0°) and side (90°) fluorescence were detected for each spermatozoon, and each detection was represented as an event on a dot plot. Correctly oriented spermatozoa were gated within the R1 region (Fig. 1a). Orientation occurred when the surface of the spermatozoon's head simultaneously faced the laser and the 0° forward detector, with the lengthwise dimension of the spermatozoon aligned towards the 90° detector. Region R5 represented all of the spermatozoa that incorporated the food dye (FDA), which were considered non-viable spermatozoa (Fig. 1a). The remaining spermatozoa not included in the R1 and R5 regions were either understained or incorrectly oriented. The X-chromosome-bearing spermatozoa (R6) were selected by gating the R1 region (Fig. 1b). The first parameter evaluated was the ability to split a population, and only samples that resolved into two populations, representing X-and Y-chromosome-bearing spermatozoa (Fig. 1c), were analysed for sorting efficiency. In these samples, the percentages of non-viable spermatozoa (FDA+), oriented spermatozoa (OS), selected spermatozoa (SS) and the average sorting rates (SR, sorted spermatozoa/s) were analysed to determine the sorting efficiency.

image

Figure 1. Dot plot and histogram created by SUMMIT computer software after the detection of the forward (0°) and side (90°) fluorescence of Hoechst 33342-stained spermatozoa. (a) Region R1 represents the percentage of correctly oriented canine spermatozoa. Region R5 represents spermatozoa stained with FDA (FDA+) that were selected as dead. (b) Region R6 represents fluorescent signals from X-chromosome-bearing spermatozoa gated in the R1 region. (c) Flow cytometric histogram outputs showing two peaks corresponding to fluorescence signals generated by canine X- and Y-chromosome-bearing spermatozoa

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The X-chromosome-bearing sperm population was selected and collected into 50-ml sterile polypropylene tubes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) pre-coated with BSA (1% in PBS w:v) and containing 2.5 ml of TCF-EY Ext. A total of 20 × 106 sorted spermatozoa were collected per tube. After sorting, the samples of spermatozoa were pelleted (3000 × g for 4 min; Megafuge 1.0 R; Heraeus), and the supernatant was removed. The resultant pellet was rediluted in TCF-EY Ext to a concentration of approximately 20 × 106 spermatozoa/ml for the evaluation of the sperm quality. Re-analysis of the sorted samples was performed to assess the accuracy of the sorting. For re-analysis, an aliquot of the X-chromosome-sorted samples containing a minimum of 5 × 106 spermatozoa was restained with additional H-42 to determine the proportion of X-chromosome-bearing spermatozoa (Welch and Johnson 1999).

Spermatozoa quality assessment

The motility of spermatozoa was evaluated objectively using a computer-assisted analysis system (ISAS; Proiser R+D, Paterna, Spain). The samples were analysed at a concentration of 20 × 106 spermatozoa/ml. For each evaluation, a 5 μl aliquot of each sample was placed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) that was pre-warmed to 38°C. At minimum, 200 spermatozoa per sample were analysed. Before the track sequence was analysed, the trajectory of each spermatozoon was identified and recorded in each field, and each was assessed visually to eliminate possible debris and to decrease the risk of including unclear tracks in the analysis. The sperm motility variable recorded was the overall percentage of motile spermatozoa [total motility, (TM)]. Spermatozoa with an average path velocity (VAP) ≥ 20 μm/s were considered motile, in accordance with the parameters provided by the manufacturer.

Sperm viability was evaluated by simultaneous cytometric assessment of the plasma and acrosomal membrane integrity using a triple-fluorescence procedure, as described previously by Martínez-Alborcia et al. (2012) and modified for canine spermatozoa. Briefly, 1.5–2 × 106 spermatozoa were transferred to tubes containing 50 μl of TFC-Ext, 4 μl of H-42 (0.05 mg/ml in PBS), 2 μl of propidium iodide (PI, 0.5 mg/ml in PBS; Molecular Probes Europe BV, Leiden, the Netherlands) and 2 μl of fluorescein-conjugated peanut agglutinin (PNA-FITC, 200 μg/ml in PBS). The samples were incubated at 38°C in the dark for 10 min. Immediately prior to analysis by flow cytometry, 400 μl of TCF-Ext was added to each sample. The fluorescence spectra of H-42, PI and PNA-FITC were detected using a 450/50 nm band-pass (BP) filter, a 670 nm long-pass (LP) filter and a 530/30 nm BP filter, respectively. Flow cytometry analyses were performed at room temperature under dimmed light using a BD FACSCanto II flow cytometer (Becton Dickinson & Company, Franklin Lakes, NJ, USA) equipped with three lasers as excitation sources: blue (488 nm, air-cooled, 20 mW solid state), red (633 nm, 17 mW HeNe) and violet (405 nm, 30 mW solid state). Data were acquired using BD FACSDiva Software (Becton Dickinson & Company). Non-spermatozoon events were gated out based on their H-42 fluorescence (DNA content), and acquisitions were stopped after 10 000 H-42-positive events. Only results corresponding to viable spermatozoa (intact plasma and acrosomal membranes; PI−/PNA-FITC−) or to spermatozoa showing damaged/reacted acrosomes (PNA-FITC+) were included in the results.

Experimental design

In this experiment, 18 SRFs from six dogs (three SRFs per dog) were used. After collection, the SRFs were processed for sperm sex-sorting as follows: the samples were split into four aliquots of 1 ml of diluted semen each, containing 100 × 106 spermatozoa/ml. One aliquot each was stained with 5, 7.5, 10 and 12.5 μl of H-42 stock solution [5 mg/ml (9 μm); w/vol in bidestilated water], for a total of four experimental groups per ejaculate. Final H-42 concentration in each aliquot was 0.045 0.067, 0.09 and 0.11 μm for 5, 7.5, 10 and 12.5 μl of H-42 experimental groups, respectively.

The values for TM, percentage of viable spermatozoa and percentage of spermatozoa with damaged/reacted acrosomes were recorded in fresh spermatozoa (washed once and studied before the staining process), spermatozoa studied immediately after the sorting procedure (AS; evaluated at approximately 1 × 106 spermatozoa/ml) and spermatozoa studied after a centrifugation step (AC; evaluated at 20 × 106 spermatozoa/ml).

Statistical analysis

All analyses were performed using the IBM SPSS statistics package (SPSS Inc., Chicago, IL, USA), version 19. In the light of the normal distribution of the data, an anova was used to analyse various parameters. A two-factor mixed factorial design was applied with interactions, including the identity of the male sample donor and the concentration of H-42 staining, used in each evaluated sorting parameter. Values reported are expressed as the least square means (LSM) ± the standard error of the mean (SEM), and statistical significance was considered at p < 0.05.

Results

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

The mean concentration of spermatozoa in the ejaculate was 497.6 ± 167.37 × 106 spermatozoa/ml. The mean values for TM, the percentage of viable spermatozoa and the percentage of spermatozoa with damage/reacted acrosomes in fresh semen (SRFs washed and analysed before the staining process) were 87.84 ± 1.4%, 91.29 ± 1.4% and 2.58 ± 1.82%, respectively.

The ability to produce a chromosome-sorted sample was significantly influenced by H-42 staining (p < 0.01) but not by the male sample donor. X- and Y-chromosome-bearing spermatozoa populations were identified in 77.5 of the samples stained with 5 μl of H-42, while all of the samples stained with 7.5, 10 and 12.5 μl of H-42 resolved into two populations clearly representing X- and Y-chromosome-bearing spermatozoa.

Because 5 μl of H-42 stain was significantly weaker (p < 0.05) in its ability to produce a split than the other concentrations tested, only the values for sorting parameters corresponding to 7.5, 10 and 12.5 μl H-42 stains are shown in Table 1. All of the parameters evaluated, such as the incorporation of the FDA dye by spermatozoa (% FDA+), the percentage of correctly oriented spermatozoa (OS), the percentage of separated spermatozoa (SS) and sorting rates (SR), were significantly influenced by the identity of the male sample donor (p < 0.01) but not by the concentration of H-42 used. In all of the cases studied, regardless of the identity of the male sample donor and the H-42 concentration used, the mean value of the sorting accuracy for the separated X-chromosome-bearing spermatozoa populations was within 85–90%. Spermatozoa quality, assessed in sorted spermatozoa obtained from 7.5, 10 and 12.5 μl H-42-stained samples, was not significantly affected by the sex-sorting process. The values of TM (Fig. 2a) and percentage of viable spermatozoa (Fig. 2b) were similar in the fresh and after-sorting (AS) samples, with the values for both parameters being between 85 and 90%. However, after the centrifugation step (AC), the percentages of TM and of viable spermatozoa were significantly lower than those obtained from the fresh and AS samples, (p < 0.01), ranging from 61% to 72% and from 69% to 75%, respectively. These differences were observed in all of the samples evaluated. Similarly, the percentage of spermatozoa with damaged/reacted acrosomes increased significantly (p < 0.01) in the AC samples, ranging from 13% to 21%, compared with the values obtained from the fresh and AS samples, which ranged from 1% to 3.5%.

image

Figure 2. Percentages (LSM ± SEM) of TM (total sperm motility) (a) and viable spermatozoa (b) in fresh, after sorting (AS) and after centrifugation (AC) samples.a,b Different superscripts indicate differences (p < 0.01) between samples at different steps of the evaluation

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Table 1. Parameters of sorting (expressed as LSM ± SEM) evaluated in samples from different dogs stained with 7.5, 10 and 12.5 μl of H-42
MaleH-42 Stain (μl)FDA+ (%)OS (%)SS (%)SR (spermatozoa/s)
  1. FDA+, percentage of non-viable sperm; OS, percentage of correctly oriented spermatozoa; SS, percentage of X-chromosome-bearing separated spermatozoa; SR (spermatozoa/s): Sorting Rates, average number of sperm sorted per second.

17.510.0 ± 3.237.6 ± 2.341.5 ± 2.03400 ± 200
109.6 ± 3.240.0 ± 2.340.9 ± 2.03800 ± 200
12.511.6 ± 3.238.8 ± 2.337.3 ± 2.03100 ± 200
27.528.3 ± 3.227.8 ± 2.334.8 ± 2.02900 ± 200
1030.2 ± 3.226.1 ± 2.334.1 ± 2.03000 ± 200
12.528.7 ± 3.228.4 ± 2.336.0 ± 2.03100 ± 200
37.58.2 ± 3.245.7 ± 2.340.0 ± 1.94300 ± 200
109.6 ± 3.245.2 ± 2.338.1 ± 1.94400 ± 200
12.510.8 ± 3.243.6 ± 2.337.7 ± 1.94000 ± 200
47.510.1 ± 3.243.3 ± 2.343.1 ± 1.94600 ± 200
1012.1 ± 3.241.0 ± 2.344.4 ± 1.94500 ± 200
12.58.0 ± 3.240.4 ± 2.747.2 ± 2.44600 ± 200
57.522.5 ± 3.227.9 ± 2.329.6 ± 1.92600 ± 200
1021.9 ± 3.229.7 ±  2.334.1 ± 1.92800 ± 200
12.529.6 ± 3.928.4 ± 2.829.9 ± 2.42100 ± 300
67.59.3 ± 3.236.9 ± 2.342.4 ± 1.93500 ± 200
108.5 ± 3.239.0 ± 2.342.7 ± 1.93800 ± 200
12.58.4 ± 3.237.0 ± 2.338.4 ± 1.93200 ± 200
ProbabilityMale0.0000.0000.0000.000
H-42 StainNSNSNSNS
InteractionNSNSNSNS

Discussion

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

To the best of our knowledge, this is the first study of the design of an optimal H-42 staining protocol for flow cytometric sperm sex-sorting in dogs. In addition, this study provides novel information about the effects of H-42 concentrations and the identity of the male sample donor on sex-sorting parameters, as well as relevant data regarding the quality of canine spermatozoa after sorting process.

The difference in DNA content between X- and Y-chromosome-bearing canine spermatozoa has been established at 3.9% (reviewed by O′Brien et al. 2009). After the establishment of this parameter, one of the basic parameters for obtaining an optimal sorting output when this technology is applied to a new specie is to establish the minimum effective H-42 concentration (reviewed by Maxwell et al. 2004), which was one of the objectives of this paper. In this regard, our results have demonstrated a lower resolution (77.5%) for X- and Y-chromosome-bearing spermatozoa populations for samples stained with 5 μl of H-42, while higher concentrations of the fluorochrome (7.5, 10 and 12.5 μl) resulted in a better splitting of the population, with 100% of the samples stained with these higher concentrations showing optimal differentiation of X- and Y-chromosome-bearing spermatozoa, regardless of the identity of the individual sample analysed. Aspects related to the highly condensed chromatin of canine spermatozoa (García-Macias et al. 2006) likely render the binding of H-42 to DNA difficult, explaining why a lower H-42 dosage leads to a lessened ability to resolve the X- and Y-chromosome-bearing spermatozoa populations. Factors such as staining time, temperature and the extender used can also influence staining efficiency. It has been demonstrated that the fluorochrome becomes more hydrophobic at a higher pH, increasing its diffusion across the membrane of a spermatozoon and yielding more efficient staining at pH 7.4 (Garner 2009). Further studies should be conducted to determine whether the use of more alkaline extenders could optimize staining protocols for canine spermatozoa by reducing H-42 stain concentrations and/or incubation times.

Our results demonstrated that all of the sorting parameters evaluated were significantly influenced by the donor of the sample, but not by H-42 staining. An increase in the FDA+ percentage was reported to determine a proportional reduction in the numbers of correctly oriented stallion spermatozoa (Clulow et al. 2009), thus affecting the other sorting parameters. Similarly, results regarding the influence of ejaculate quality on spermatozoa sortability have been described for elephants and rhinoceroses (Behr et al. 2008). Therefore, the results obtained herein can most likely be explained by the fact that two of the males included in the study had spermatozoa populations with higher FDA+ values, thereby determining a lower proportion of properly oriented spermatozoa and resulting in lower sorting rates, which compromised the efficiency of the sorting process. This variability between males affecting sorting efficiency has been described previously in boars (Parrilla et al. 2005), stallions (Clulow et al. 2009) and bulls (Garner et al. 1983). This variability should be considered when selecting suitable dogs for inclusion in sex-sorting programs. Due to the great diversity of extant dog breeds, it would be interesting to conduct a study of the influence of breed on the sex-sorting process, as has been evaluated in other species, such as cattle (Garner 2006).

One remarkable finding of our study involves the percentages of correctly oriented spermatozoa, which were relatively low (ranging from 26.1% to 45.7%) as compared to those reported for other species, such as bulls (60–80%; Sharpe and Evans 2009), animals to which sex-sorting technology is applied commercially. Previous results have described low-to-moderate proportions of properly oriented spermatozoa in cats (Spinaci et al. 2007; Pope et al. 2009) and alpacas (Morton et al. 2008), and these findings have been attributed to the shapes and sizes of the heads of the spermatozoa. Garner (2006) described the tendency of spermatozoa with small, rounded or angular-shaped heads, as found in dogs, cats (Spinaci et al. 2007), horses (Gibb et al. 2011) and alpacas (Buendia et al. 2002), to be more difficult to orient in a sperm sorter.

Despite this drawback, the sorting rates obtained in our study were acceptable for those males with low FDA+ percentages (1, 3, 4 and 6), ranging from 3100 to 4600 events per second and allowing the collection of between 14 × 106 and 16 × 106 spermatozoa per hour in the best samples. This sorting efficiency is comparable to the results obtained for stallions (Behr et al. 2009), and it is better than the results obtained for other species, such as alpacas (Morton et al. 2008), cats (Pope et al. 2009), rhinoceroses (Behr et al. 2009) and elephants (Hermes et al. 2009). Because conventional AI in dogs requires approximately 200 × 106 spermatozoa per insemination (Rota et al. 2010), it is clear that these two reproductive technologies are not currently compatible. However, the insemination dose required to perform intra-uterine AI using a low numbers of sorted spermatozoa could be easier to obtain. An evidence that the combination of these two assisted reproductive technologies can provide satisfactory results is that offspring of pre-determined sex, as derived from flow-cytometrically sorted spermatozoa in combination with intra-uterine AI, was achieved in one bitch (Meyers et al. 2008).

Our results regarding the quality of spermatozoa immediately after sorting indicate that canine spermatozoa were not severely affected by the sorting process. However, the centrifugation step necessary to concentrate the highly diluted sorted spermatozoa before processing significantly affected the quality of the sorted canine spermatozoa, inducing the impairment of TM, a reduction in the percentage of viable spermatozoa and an increase in the total numbers of spermatozoa with damaged/reacted acrosomes. Although the centrifugation protocol used in this study, based on a high speed of centrifugation over a short period, was used in boars with good results (Parrilla et al. 2012), further studies should be conducted to design an adequate centrifugation protocol for sorting canine spermatozoa.

In conclusion, the sex-sorting of spermatozoa by flow cytometry can be performed successfully in canine samples using H-42 concentrations between 7.5 and 12.5 μl. Although the morphology of canine spermatozoa results in a low percentage of correctly oriented spermatozoa, acceptable results for sorting efficiency can be obtained in terms of sorting output and the quality of spermatozoa after sorting. Regardless of staining conditions, significant individual differences are evident in the sorting efficiency of samples of canine spermatozoa. Future studies should be conducted to optimize sorting efficiency in this species, to develop protocols for the preservation of sex-sorted canine spermatozoa and to allow the practical application of this reproductive technology.

Acknowledgements

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

This study was supported by MICINN (AGL2008-04127/GAN, RYC-2008-02081), University of Murcia (R-549/2009), the Seneca Foundation of Murcia (GERM04543/07), and Sexing Technologies (Texas, USA).

Author contributions

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

All authors were involved in all phases of the research and writing of this manuscript.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. References
  • Behr B, Rath D, Hildebrandt TB, Goeritz F, Blottner S, Portas TJ, Bryant BR, Sieg B, Knieriem A, De Graff SP, Maxwell WMC, Hermes R, 2008: Index of sperm sex sortability in elephant and rhinoceros. Reprod Dom Anim 44, 273277.
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