Determination of Sex Ratio In Bovine Semen Using SYBR Green Real-Time PCR

A SYBR green real-time PCR assay was developed to nd out the sex skewness in bovine sex-sorted semen samples. The qPCR assay of PLP and SRY genes revealed the mean values of X- and Y-bearing spermatozoa as 50.24 ± 0.65 and 49.75 ± 0.62 per cent in unsorted, and 91.80 ± 0.79 and 8.20 ± 0.73 per cent in X-enriched semen samples respectively.. The amplication eciencies of the PLP and SRY primers were 99.25 and 98.03 per cent respectively. The method was validated by a series of repeatability and reproducibility assays which revealed low co-ecients of variations as 2.19 and 3.12 per cent respectively Thus becoming a reliable and inexpensive tool to evaluate the sorted semen on routine basis and validation of other sperm sexing technologies.


Introduction
The main aim of a breeder is to increase the genetic potential to the maximum level and to produce a desired off-spring under optimal conditions (Pindaru et al., 2016). The scienti c approach for the preselection of the sex of off-spring was started in 20th century (Seidel, 2012). Among several conventional techniques such as albumin gradient, swim-up, percoll density gradient, free ow electrophoresis, H-Y antigen and sorting based on volumetric differences, ow cytometry effectively sorted the X-and Ychromosome bearing spermatozoa. This method is mainly based on the fact that X-bearing (female) spermatozoa contains 3.8 per cent more DNA than Y-bearing sperm (Johnson, 2000). Flow cytometric separation of spermatozoa was rst done by Johnson et al. (1989) in rabbits. Later the technique was used successfully in several species (Johnson, 2000;Seidel and Garner, 2002;Maxwell et al., 2004;Lu et al., 2007;Liang et al., 2008). The e ciency and success of arti cial insemination with sorted semen depends on the accuracy of sexing of the spermatozoa (Pindaru et al., 2016) and high sorting accuracy is the pre-requisite for wide spread use of sexed semen (Habermann et al., 2005). The purity of sexed semen is usually veri ed by ow cytometric reanalysis of the DNA content of semen (Welch and Johnson, 1999). But there is a need for a reliable method, which does not depend on the same instrumentation to prevent the inherent errors of poorly sorted semen (Colley et al., 2008).
Speci c DNA sequences on X-and Y-spermatozoa enable the identi cation of sex of individual sperm and sex ratio of semen samples (Colley et al., 2008;Maya et al., 2013). Several methods were developed to determine sperm sex ratio such as duplex PCR (Colley et al., 2008) or single cell PCR (Yadav et al., 2017), but these techniques were associated with a disadvantage of requirement of large number of individual sperms (Maleki et al., 2013;Yadav et al., 2017). Quantitative real-time PCR (qPCR) technique emerged subsequently with some advanced features for the sex determination of spermatozoa more simply and accurately in pooled semen samples (Maleki et al., 2013). Quantitative PCR allows simultaneous ampli cation of the DNA and real time monitoring of amount of ampli ed DNA through reaction cycles. Therefore, the present study was aimed at development and validation of SYBR green real-time PCR assay for the quanti cation of X and Y-bearing spermatozoa in bovine semen samples.

Materials And Methods
Semen samples and sperm DNA extraction A total of seven unsorted and four X-enriched frozen semen samples were thawed and washed twice with phosphate buffered saline. The sperm pellet was resuspended in 2.5 ml of sperm lysis buffer (50 µl of 1M Tris-HCl, 100 µl of 0.5M EDTA, 100 µl of 5M NaCl, 0.1 g of 4% SDS and 50 µl of 2% Mercaptoethanol) and 2.2 ml of nuclease free water by vortexing and incubated for 20 minutes at 56 ºC in a water bath. To the suspension, DNAzol (Thermo Scienti c, USA) was added, mixed thoroughly and incubated for 2-3 minutes at room temperature. Later, Phenol:Chloroform:Isoamylalcohol (25:24:1) was added, mixed well by vortexing and centrifuged at 11,000 rpm for 10 minutes. The aqueous phase was collected into a separate 2ml eppendorf tube, added 24:1 ratio of chloroform:isoamylalcohol and mixed throughly by vortexing. After centrifuging at 11,000 rpm for 10 minutes, aqueous phase was collected into a separate tube, equal volume of isopropanol added, centrifuged and the supernatant discarded. Later, the DNA pellet was treated with 70 per cent and absolute alcohol, air-dried and resuspended in 50 µl of TE buffer. The purity and concentration of DNA samples were estimated by gel electrophoresis and spectrophotometry (NanoDrop OneC of Thermo Scienti c, USA).

Primers
Gene speci c nucleotide sequence was obtained from NCBI for PLP gene (Genbank Accession No. AJ009913.1) and cattle X-speci c PLP primers were designed using Primer-BLAST software. The forward and reverse primer sequences for PLP gene were 3'-ACC AAG GGA AGA GCA GGA AT-5' and 3'-TGC CAA CTT GTA CCC AAA CA-5' respectively. The primers for Y-speci c SRY (Sex determining region Y) gene was synthesized as described by Hamilton et al. (2009). PCR ampli cation was carried out using thermal cycler (Applied Biosystems 2720).

Sybr Green Real-time Assay
The quantitative PCR was performed with 5 µl of SYBR green mix (Sso A dvanced Universal SYBR Green super mix, Bio-Rad), 3 pmol each of forward primer and reverse primers (0.3 µl), 70 ng of sperm DNA as template (1 µl) and the nal volume of reaction was made up to 10 µl by nuclease free water. The ampli cation pro le consisted of initial denaturation at 95˚C for 2 minutes, denaturation at 94 ˚C for 30 seconds, annealing at 64˚C for 30 seconds, extension at 72˚C for 30 seconds and melt curve at 72-98˚C with every 0.2ºC hold for 1 second. The ampli ed PCR products were checked on 2 per cent agarose gel with 100 bp DNA ladder. The bands were viewed in a GelDoc (Bio-Rad Laboratories Inc., USA) system and the images stored.
Generation Of Standard Curve PLP and SRY genes were ampli ed in a 50 µl PCR reaction mixture. PCR products were resolved with 2 per cent agarose gel and bands were sliced under UV illumination. The bands were puri ed from gel with Favrogen gel/PCR puri cation kit (Favorgen, Taiwan). The concentration of puri ed PCR products was measured using NanoDrop. The speci city of the PCR product was rechecked with 2% agarose gel. Using concentration of DNA and product length, copy number per µl volume was calculated with online calculator for determining the number of copies of a template (URI Genomics and Sequencing Centre).
The concentration of the stock solution was adjusted to 1 X 10 6 copies. Then, the stock solution was diluted serially by 10 folds to obtain a standard series from 1 X 10 6 to 1 X 10 2 copies. A standard curve was constructed with different copy numbers in triplicates. The C T values obtained during ampli cation of each dilution were plotted against logarithm of their template dilution factor. The coe cient of determination (R 2 ) value obtained from the equation of linear regression line was used to evaluate the qPCR aasay. Copy numbers for each gene was calculated separately and converted into percentages by using the formula suggested by Parati et al. (2006). A Chi-square test was used to determine whether the observed percentages of X-and Y-spermatozoa in a semen samples differed signi cantly from expected sex ratio 1:1. The data were analyzed with IBM SPSS Statistics 20 software.

Primer speci city analysis
The primers used for PLP and SRY genes showed uniqueness in amplifying a single PCR product in real time assay as there was neither primer dimer nor non-speci c products in reaction. Further analysis by agarose gel con rmed the melt curve analysis. None of the control yielded any signal prior to 30 cycles.
Both the primers showed a single melting peak at temperatures 82.20 ºC (PLP) and 84.40 ºC (SRY) as shown in Fig. 1.

Standard curve
The standard curves obtained showed a linear relationship (R 2 =0.99) between logarithm of dilution factors and Ct values for serial template dilutions (Fig. 2). The Ct values ranged from 8.55 (1x10 8 ) to 27.34 (1x10 2 ) for PLP gene and 10.38 (1x10 8 ) to 28.59 (1x10 2 ) for SRY gene ( Table 1). The end point of qPCR revealed a clear distinct band in decreasing intensity as a result of serial dilution of the amplicons without formation of any multiple non-speci c ampli cation (Fig. 3).

Repeatability And Reproducibility Assay
The mean co-e cient of variations resulted from the repeatability and reproducibility assays were 1.45 and 2.69 respectively (  The mean values for X-and Y-bearing spermatozoa in X-enriched semen samples were 91.80 ± 0.79 and 8.20 ± 0.73 per cent, ranging from 89.72 to 94.37 and 5.63 to 10.28 respectively (Table 3). While the Chisquare statistical test of goodness of t revealed a signi cant difference (P<0.01) between the X-and Ybearing spermatozoa in the X-enriched semen samples and no signi cant difference between the observed and expected per cent of X-and Y-spermatozoa in unsorted semen samples with a mean sex ratio equal to 1:1 (50.24 : 49.75) across all the tested unsorted semen samples.
Real-time quantitative PCR for determination of sperm sex ratio carried out in this study gave an accurate result. PLP and SRY genes were used as gender markers for identi cation of X-and Y-bearing spermatozoa respectively. Both the genes are present in single copy on X-and Y-chromosomes, therefore, every single copy of PLP and SRY sequence detected indicated the presence of respective quantities of Xand Y-bearing spermatozoa (Tan et al., 2015).
Since all gene sequences for a given species are represented, genomic DNA could serve as universal standard for the absolute quanti cation of any expressed gene (Yan et al., 2006). In this study, puri ed PCR products of PLP and SRY genes were used to generate the standard curves. Both the genes gave different Ct values with same gene copy numbers due to differences in the length of gene fragments (Tan et al., 2015). Therefore, two different standard curves were obtained for quanti cation of X-and Ychromatin bearing spermatozoa. Lower the mean values of co-e cient of variation for repeatability and reproducibility, higher would be reliability of this approach in absolute quanti cation of X-and Ychromatin contents in semen samples.
From the copy numbers of PLP and SRY genes, the quantity of X-(50.24%) and Y-(49.75%) chromosome bearing spermatozoa did not differ from 1:1 ratio, which is reported as normal in unsexed mammalian semen samples (  To conclude, the sex determination in bovine unsorted semen samples was performed successfully with SYBR Green uorescent dye. The SYBR Green real time quantitative PCR is a rapid and reliable technique in absolute quanti cation of sex ratio in bovine semen samples. This method is a valid tool for routine validation of high number of sorted semen samples, determination of sex ratio in pooled semen samples and can also be used for the validation or calibration of other related techniques.

Statements And Declarations
Acknowledgements: The nancial support provided by Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai is gratefully acknowledged.
Funding information: The authors declare that no funds and grants were received during the preparation of this manuscript.
Con ict of interest: The authors have no relevant nancial or non-nancial interests to disclose.