QUICK CYTOGENETIC SCREENING OF BREEDING BULLS USING FLOW CYTOMETRIC SPERM DNA HISTOGRAM ANALYSIS

Department of Animal Sciences and Animal Husbandry, Georgikon Faculty, University of Pannonia, Deák F. u. 16, H-8360 Keszthely, Hungary; Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Saari Unit, Saarentaus, Finland; Institute of Animal Science, Biotechnology and Nature Conservation, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Debrecen, Hungary

According to Amann and Hammerstedt (1993), the fertilising spermatozoon has to have a series of attributes in order to achieve its goal: acceptable morphology, proper metabolism for energy production, progressive motility, capacity for hyperactive motility, etc.In addition, the genetic package of the fertilising spermatozoon must contain genes needed for development and lack lethal mutations or extra genetic material preventing development.It can be hypothesised that the higher the proportion of motile, viable, morphologically normal spermatozoa with intact chromatin in the insemination dose, the better the chances for pregnancy (Morrell and Rodriguez-Martinez, 2009).Although motility and viability (and less frequently morphology) are routinely assessed in AI stations, the status of the genetic material is rarely tested.
The status of the paternal genome can be viewed at three stages: besides the increasing focus on genomic selection of breeding males (Amann and DeJarnette, 2012), the intactness of the chromatin and the haploid set of chromosomes Acta Veterinaria Hungarica 64, 2016 in the spermatozoon are different levels where we can assess the abovementioned status.The organisation of the sperm chromatin is unique, as histone proteins are eventually replaced by transition proteins and finally by protamines, resulting in an extremely compact, condensed DNA (Dadoune, 1995;Sakkas et al., 1999).Proper condensation may stabilise the DNA and makes it less sensitive to oxidative damage; however, mature spermatozoa are not able to repair DNA damage.Abnormalities of the sperm chromatin structure can cause disturbances in fertility, pronuclear formation, early embryo quality and pregnancy outcome.These abnormalities are classified as 'uncompensable', as subfertile males cannot be brought to normal fertility by increasing the inseminate dosage.Sperm cells with uncompensable defects are able to fertilise oocytes; however, embryo development may be abnormal (Evenson, 1999).
The problem with paternal chromosomes can be viewed at two levels: (a) structural (i.e.translocations, deletions, duplications, etc.), and (b) numerical (alterations in the normal, haploid chromosome numbers).
Since the 1960s, animal cytogenetics has become more and more important (Ducos et al., 2008).The cytogenetic quality of breeding bulls and boars has great economic importance (Larsen et al., 2004).Classic techniques to detect abnormal karyotypes are based on microscopic analysis of peripheral blood lymphocytes (Matsson et al., 1986).Microscopic countings, however, are time consuming and require specialised expertise.Flow cytometry offers a more precise and quick alternative, and it was successfully applied in karyotyping of several mammalian species including sheep, cattle, pig (Dixon et al., 1992), dog (Langford et al., 1996) and human (van den Engh et al., 1985).Flow karyotyping has not become a routine evaluation for domestic animals as the technique is rather difficult and the resolution of bivariate flow karyograms is usually not perfect, i.e. the smaller chromosomes are not distinguishable.Moreover, individual chromosome polymorphisms can result in differing flow karyotypes between healthy individuals.The sensitivity of the technique stands between that of traditional light microscopic chromosome banding and flow cytometric assessment of the total DNA content of whole cells (Givan, 2001).
Flow cytometry offers a tool to measure the total DNA content of sperm cells (Lewalski et al., 1991), which has several advantages, like no need to collect blood and the possibility of monitoring transported semen without actually meeting the animal in question.Translocation carriers were successfully detected by flow cytometry (Lewalski et al., 1991(Lewalski et al., , 1993)).The flow cytometric DNA content analysis of spermatozoa has led to the successful separation of X-and Ychromosome bearing germ cells (Gledhill et al., 1976;Meistrich et al., 1978).The unique paddle-like shape of the mammalian spermatozoon, however, is a great technical challenge, and special hydrodynamic cell orientation is needed to ensure that every spermatozoon will have the same orientation allowing the fluorescence measurement of the flat surface of the cells (Garner, 2001).In our pre-vious studies (Revay et al., 2009(Revay et al., , 2010) ) our flow cytometric analyses revealed spermatozoa with abnormal chromosomal content and incomplete chromatin condensation.
To overcome the need of a sophisticated flow cytometer equipped with a sperm head orienting device, Larsen et al. (2004) developed a simplified approach for the detection of males with spermatozoa carrying cytogenetic aberrations.They applied a series of enzymatic and chemical treatments for sperm head decondensation to have swollen and rounded sperm nuclei, eliminating the need of sperm orientation.They developed a model for normality of the sperm DNA histograms: two subpopulations (i.e.X-and Y-chromosome bearing germ cells) and histogram CV equal to or less than 1.3%.They stated, however, that their protocol was unfeasible in its current form for routine screening purposes.They suggested several critical points for the improvement; however, these have not been carried out so far.
Recently, relatively cheap and easy-to-use benchtop flow cytometers have become available even for the use in routine AI centres for daily semen quality control (Hossain et al., 2011).Most of these instruments are, however, equipped with a 488 nm laser which is not suitable for the DNA-specific fluorochromes used in sperm sexing and other quantitative DNA analyses, namely the Hoechst dyes and 4'6-diamidino-2-phenylindole (DAPI) stain.
The aim of the present study was to test another fluorochrome which is properly excited with the 488 nm laser line and therefore would be suitable for routine DNA analyses aiming to detect breeding males and/or insemination doses carrying cytogenetic aberrations.In a series of experiments we first established basic DNA histogram parameters of cytogenetically healthy breeding bulls by measuring the intraspecific genome size variation of three animals (each of them three times -as suggested by Dolezel and Bartos, 2005), then we compared the histogram profiles of bulls carrying cytogenetic defects to the baseline values.

Semen samples
In Experiment 1 we used frozen-thawed semen samples from three bulls free from cytogenetic defects.In Experiment 2 one of the bulls from Experiment 1 was used as control (bull A) and bulls with the following cytogenetic aberrations were tested: bull B was a carrier of 2;4 reciprocal translocation (Switonski et al., 2008); bull C was a carrier of 20;24 reciprocal translocation (Andersson et al., 1992); bull D produced approximately 20-25% diploid spermatozoa (Revay et al., 2010); bull E had a translocation between chromosomes 6 and 29 (Venhoranta et al., 2013).

DNA labelling
Sperm DNA was labelled with propidium iodide (PI) as part of the FXCycle PI/RNase kit (F10797, Molecular Probes).
Labelling followed the protocol suggested by the manufacturer: spermatozoa were fixed in 70% ethanol, then washed from the fixative (centrifuged at 400 × g for 10 min).Five hundred µl of FXCycle PI/RNase staining solution was added to the cell pellets (approximately 1 × 10 6 /ml spermatozoa) and after vortexing the samples were incubated for 30 min at room temperature in dark before flow cytometric analyses.

Flow cytometry
We measured the DNA quantity expressed as PI fluorescence intensity with a Beckman Coulter FC500 flow cytometer, equipped with a 488 nm 20 mW argon ion laser.PI fluorescence was detected on an FL3 detector (655 nm LP) in linear mode.Sperm head orientation was not taken into consideration as our benchtop flow cytometer was not equipped with such an orienting device.Doublet discrimination was done according to the suggestions of the manufacturer (Beckman Coulter).Five thousand events per sample were recorded and scatter and fluorescence intensities were stored in list mode files.The flow cytometer was calibrated daily with FlowCheck fluorospheres (6605359, Beckman Coulter); moreover, in order to monitor random drift in fluorescence intensities between samples, every sample contained 10 µl FlowSet fluorospheres (6607007, Beckman Coulter) serving as internal control.

Analysis
In Experiment 1, one-parameter PI fluorescence intensity histograms were drawn and individual histogram profiles were compared with the Kolmogorov-Smirnov option in the CXP Analysis software (Beckman Coulter) of the cytometer.This analysis assesses the difference between two populations and shows the maximum difference between two cumulative frequency distributions expressed as Dmax value (Young, 1977;Watson, 2001).Dmax values were calculated for paired histograms (three bulls, three repeats: n = 36 pairs) and these values were checked for normality using Lilliefors test and descriptive statistics were established with Statistica for Windows (version 8, Statsoft Inc.).
In Experiment 2, the histogram profiles (Fig. 1) of the carrier bulls were compared to the control bull using the Kolmogorov-Smirnov test as described above.The threshold Dmax value to differentiate healthy and possibly defective animals was derived from the descriptive statistics in Experiment 1 and was calculated as mean + 2SD (Indrayan, 2012).

Results
Experiment 1 Dmax values of the paired histograms are shown in Table 1.The Kolmogorov-Smirnov test revealed significant differences between bulls or even between repeated measurements of the same individual (P < 0.05) with the exception of bull 2 where the repeated measurements were not significantly different.However, since the Kolmogorov-Smirnov test is reported to be oversensitive (Parikh et al., 1999;Lampariello, 2000) we did not consider this as a flaw of the data analysis approach.

Experiment 2
The histogram profiles (Fig. 1) of the carrier bulls differed significantly (P < 0.01) from the control bull (A) and the Dmax values were as follow: bull A vs. B: 0.245; A vs. C: 0.128; A vs. D: 0.416; A vs. E: 0.166.With the exception of bull C, every carrier bull showed higher Dmax value than the threshold value established in Experiment 1.This can be explained as in that case smaller chromosomes (chromosomes 20 and 24) were involved, so probably only a very small amount of DNA was missing or added in the affected sperm cells.

Discussion
In our opinion this flow cytometric approach has several advantages.The fluorescent labelling kit is easy to use, does not require excessive sample preparation, quick, and the fluorochrome can be analysed with the standard laser line and optics of every benchtop flow cytometer.The Kolmogorov-Smirnov option is available in the software of most flow cytometer brands, therefore no additional software purchase is needed.Individual bulls or even insemination doses can be evaluated quickly, so the method can be applied to test the animal even if he is not physically present (like in the case of semen import).We suggest that every laboratory should establish its threshold values following the study design described above as flow cytometer instruments with different optical settings may result in different Dmax values; moreover, different cattle breeds (due to Y chromosome polymorphisms, for example) may have different DNA histogram profiles.The test fits into the laboratory routine where flow cytometry is applied for semen quality control, and this analysis should be carried out only once in the lifetime of a bull.

Fig. 1 .
Fig. 1.DNA histogram overlays of the carrier bulls (B-E; white histograms) and the control bull (A; grey).X-axis shows PI fluorescence, expressed on a linear, 1023-channel scale

Table 1
Matrix of Dmax values of paired histograms of the three healthy bulls measured in three repeats