Five Layered Optiprep based Density Gradient Model is a Promising Model for Enrichment of Viable X Chromosome Bearing Spermatozoa in Bubalus bubalis

It is essential to maintain higher ratio of elite female to male for sustainability of the dairy sector. In addition to the faster genetic progress obtained through use of sex sorted sperm or embryos, there are also additional advantages for the management and efficiency of livestock production. There has been great interest in sex selection in the bovine and there are clear economic and management advantages to be gained (Kumar et al., 2016). This can be achieved by effective sorting of X (female) or Y (male) chromosome-bearing spermatozoa in semen. Amongst various experimental techniques for separation of X and Y sperm, (Prakash et al., 2014), flow cytometry method which is based on DNA differences poses to be a major breakthrough in reproduction technology Blondin et al., (2009). This technique was developed by Johnson et al.,1989, at the USDA Beltsville Agricultural Research Center and is the only method validated so far for sex selection before birth (Graaf et al., 2009; Prakash et al., 2014).  But, in addition to being a costly technique, many studies have reported that sperms sexed using this technique possess reduced fertility (Seidel et al., 1999; Sartori et al., 2004; Seidel and Schenk, 2008).                                           
       
 A simple methodology of density gradient centrifugation has been found to be capable of separating X and Y chromosome bearing spermatozoa with lower cost and without damages to sperm viability in bovines. Over the years, variety of different compounds has been developed as density gradient media in order to enhance the separation process. Percoll gradient density has been used extensively and successfully for spermatozoa selection (Promthep et al., 2016). Concerns have also been raised regarding Percoll’s safety and its use in assisted reproductive technologies (Hossepian et al., 2000). Sucrose as a low cost alternative was used for density gradient media but with limited success Kanesharatnam et al., (2012). Therefore, the primary objective of this study was to develop appropriate density gradient model for enriching viable X chromosome bearing spermatozoa population in semen of Murrah buffalo bull. A convenient, precise and accurate procedure to identify X- and Y-sperm is essential for validating the accuracy of sperm sexing. Since, real time PCR analysis based on fluorescence DNA dyes such as SYBR green offers a quick, simple, cheap, independent, pooled semen sex ratio determination approach, (Parati et al., 2006; Maleki et al., 2013) the presumptive X or Y chromosome bearing sperm population in enriched semen was evaluated using SYBR green based real time PCR.
       
Considering that sperms enriched through density gradient centrifugation are submitted to a variety of adverse conditions such as movement across layers of different densities of media and centrifugal force, in vitro evaluation for sperm quality was performed by assessment of motility, plasma membrane integrity and DNA integrity (Amann and Hammerstedt, 1993). Therefore, present study was proposed with objective of developing appropriate density gradient model for enriching X chromosome bearing spermatozoa population in unfrozen extended semen of buffalo bull, determination of sex ratio of X and Y chromosome bearing spermatozoa in enriched semen by SYBR green based real time PCR and evaluation for viability of semen processed through different density gradient media.

The study was conducted at Reproductive Biotechnology Laboratory of Department of Animal Biotechnology situated at Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana from 2015- 2018.
 
Collection of semen samples
       
Semen samples were collected from proven elite bulls of Murrah buffalo, at semen bank of Haryana livestock development board (HLDB), Hisar, Haryana using artificial vagina method. Semen samples were examined under light microscope for assessing mass and individual motility and the samples with higher than 80% progressive motility were selected for the study. The selected ejaculates from bulls were extended with egg yolk phosphate diluter to make the final concentration of 20 million sperms / 220 µl of semen and were pooled in equal quantities at each collection time to eliminate individual variation.
 
Preparation of density gradient models for enrichment of X spermatozoa population
 
Four gradient media viz., Percoll, Optiprep, Ficoll and Sucrose were used for preparation of DGC models. All density gradient models were prepared by standard technique described below.
       
Every DGC model preparation involved layering 1 ml of successive decreasing densities of media solution upon one another in 15 ml polystyrene centrifuge tubes. On the top i.e., over the uppermost layer, 1 ml of semen with concentration of 2 x10⁶ sperms /220µl, was layered. Description regarding use of various densities of media solution in different DGC models has been provided in the tables (Table 1-4). Model 1, 2 and 3 of every media were centrifuged at 200×g, for 30 minutes at 22 to 24°C and model 4, 5 and 6 of every media were centrifuged at 300×g for 30 minutes at 22 to 24°C. Immediately after the centrifugation, tube was removed from the rotor without any disturbance of the layers of density gradient models. After centrifugation, most of the supernatant was gently removed and pellet was placed into a new sterile test tube, and then pellet was again re suspended in 5 ml of HEPES buffered Hank’s solution to remove the density gradient medium. It was again centrifuged at 500×g for 7 minutes, the supernatant was removed and the final pellet was re suspended in the sterile HEPES buffered Hank’s solution to obtain final sperm concentration of 1x10⁶ /ml of medium. Finally, the suspension was centrifuged at 300xg for 10 minutes to prepare the sperm pellet. The pellet was evaluated for viability (motility and plasma membrane integrity) and processed for DNA extraction.
 

 

 

 

 
 
Establishment of discontinuous percoll gradient
 
Percoll gradients (Pharmacia Fine Chemicals, Uppsala, Sweden) were made by mixing stock solution of Percoll with DMEM, pH 7.4, 280 to 290 mOsm/Kg H₂0, with 0.3% (w/v) of BSA (Calbiochem, Darmstadt, Germany), in order to obtain densities ranging from 65% to 85%.
       
Establishment of discontinuous optiprep gradient
 
A 60% iodixanol solution in water with a density of 1.320 g/ml at room temperature is commercially available as OptiPrep (Axis-shield, Oslo, Norway). Before density gradient centrifugation, isotonic solutions of the desired densities were prepared by dilution with Hanks buffered salt solution (HBSS; 5.33 mM KCl, 0.441 mM KH₂ PO₄, 4.17 mM NaHCO₃, 137.92 mM NaCl, 0.338 mM NaH ₂ PO₄, 5.56 mM glucose, pH 7.4).
 
Establishment of discontinuous ficoll gradient
 
Density gradient media is available in commerce ready to use or ready to make the different density layers. Isotonic solutions of the desired densities were prepared by dilution with Hanks buffered salt solution (HBSS; 5.33 mM KCl, 0.441 mM KH₂ PO₄, 4.17 mM NaHCO₃, 137.92 mM NaCl, 0.338 mM NaH₂ PO₄, 5.56 mM glucose, pH 7.4).
 
Establishment of discontinuous sucrose gradient                             
Powdered sugar was placed in the oven at 60°C for 2-3 hours. Weighed sugar powder by electronic balance was dissolved in warmed 2.9% sodium citrate buffer in the beaker in order to obtain appropriate mass percent of sugar. The densest layer of sucrose (35%) was prepared by addition of 3.5g sucrose in 10 ml sodium citrate buffer solution (Kaneshratnam et al., 2012). Other layers were also prepared accordingly.
 
Swim-up procedure
 
Swim-up procedure was performed with Tyrode-albumin-lactate-pyurvate (TALP- Sigma) medium (Parrish et al., 1995). Medium was incubated in an atmosphere of 5% CO₂   in air at 38.5^°C for 2 hours prior to use. A total of 0.25 ml of semen was deposited at the bottom of 1.5 ml of medium. The tubes were incubated in an atmosphere of 5% CO₂   in air at 38.5^°C for 30 minutes and supernatant from same media tubes was pooled separately in sterile conical tube and centrifuged at 100xg for 10 minutes. The supernatant was discarded, leaving 100 µl sperm suspension at the bottom of tube. This sperm suspension was diluted with medium. This preparation was equilibrated at room temperature for 5 minutes. After adding 5 ml of more medium, it was again centrifuged for 10 minutes at 100xg. The supernatant was again discarded and remaining 100 µl of sperm suspension in each tube was diluted with same medium containing heparin (21.87 I.U. /ml) and incubated finally for 15 minutes in CO₂ incubator at 38.5^°C.
 
Simple washing of semen
 
Sodium citrate washing method (Samad et al.,1998) was used for washing of semen samples. A total of 0.25 ml of semen was mixed with 2.9% sodium citrate to make final volume of 5 ml. The suspension was centrifuged at 300xg for 10 minutes. The supernatant was discarded and the pellet containing sperms was dissolved in 3 ml of 2.9% sodium citrate and centrifuged again for 10 minutes. After discarding supernatant, the concentration was determined in sperm pellet and sufficient Tyrode solution was added to obtain final sperm concentration of 1x10⁶/ml of medium.
 
Determination of sex ratio of X and Y bearing spermatozoa in enriched semen by SYBR green based real time PCR
Primer design
 
A pair of primers specific to Bubalus bubalis X and Y-chromosome was designed sequence using primer 3 software, according to the parameters required for the SYBR green real- time PCR (Dorak, 2006). The Y-specific primers pair was designed on a conserved region of the Bubalus bubalis chromosome linked male sex determination gene (SRY) (Gen Bank accession no. JX668001.1). The Y-product amplification length was 142bp. The X- specific primers pair was designed on a conserved region of the Bubalus bubalis chromosome proteolipid protein gene (PLP) (GenBank accession no. JN182842.1). The X-product amplification length was 177bp. The set of primers are described below, Table 5.
 

 
DNA extraction from enriched semen samples
 
DNA was isolated from the final sperm pellets obtained after processing of semen through density gradient centrifugation models, swimup method and sodium citrate simple washing using Qiagen DNeasy Blood and Tissue Kit. Before proceeding to the Qiagen protocol, semen was treated with two additional buffers and proteinase K. Briefly, 200 μl of semen and 10 ml of lysis buffer (150 mM NaCl and 10 mM EDTA, pH 8.) were mixed and centrifuged at 2500 x g for 10 minutes. The pellet was resuspended in 300 μl buffer containing 100 mM Tris-Cl, pH 8.0, 10 mM EDTA, 500 mM NaCl, 1% SDS and 2% 2-mercaptoethanol and then100 μl of proteinase K was added in it. After incubation at 56°C for 2 h, another 20 μl proteinase K was added and incubated again at 56°C for 2 h (Kumari et al., 2019).  After addition of lysis buffer and ethanol in 400 μl quantities each, the mixture was applied to the mini spin column (Qiagen, Germany) and processed according to manufacturer recommendations. The quality and quantity of extracted DNA were assessed by spectrophotometer. The quality of extracted DNA was assessed by the ratio of optical density at wavelengths of 260 and 280 nm. The concentration of extracted DNA was calculated using the formula-
   
Real time PCR amplification for PLP and SRY genes
 
Real-time PCR amplifications for PLP and SRY genes from the performed in Thermal cycler (Step One Plus, Applied Biosystem) using Quantitect SYBR Green PCR kit. DNA extracted from semen enriched through density gradient models was used as template for real time PCR. The PCR mixture contained 90 ng DNA template, 5 picomoles each of forward and reverse primer, 12.5µl of q PCR Master mix. Nuclease free water was added to make the final volume of 25µl. Amplification of both genes was performed by an optimized protocol (10 min at 95°C, 35 repeated cycles of two steps at 95°C for 15 s, 60°C for 15 s and 72°C for 30s).
        
Every DNA sample was run in triplicate and each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primer dimmers by an optimized protocol. Each run included a negative control reaction.
 
Determination of gender chromosome frequencies
 
The % of X chromosome content in given semen sample was determined using the following equations; % X= {n/n+1} 100 (where n= Ct of X/Ct of Y) (Parati et al., 2006; Maleki et al., 2013).
 
Repeatability and reproducibility assays for validation
 
The repeatability (i.e., the variability of a method when repeated measures are taken with the same material in a single experiment) and reproducibility (i.e., the variability of a method when repeated measures are taken in different experiments) were assessed by computing the coefficients of variation (CV) of the X-chromosome content observed in 20 quantifications of semen samples. In particular, the repeatability assay was assessed in five runs (4 replicates per run), while 20 runs (1measure per run) were performed for the reproducibility assay.
 
Assessment of the viability of spermatozoa processed through different gradient media
Assessment of motility
 
The motility of enriched spermatozoa through different density gradient models was analyzed using the computer assisted semen analyser (CASA, Hamilton Thorn Biosciences. The concentration of semen was adjusted to 100x 10⁶ sperms/ml in normal saline. Approximately, 2 µl of the sample were placed in a 20 µl standard counting slide (Leja slides). The slide was loaded into CASA and at least 20 fields were selected for motility analysis.
 
Assessment of plasma membrane integrity                                                        
Plasma membrane integrity of sperm was assessed using hypo-osmotic swelling test (HOST). Sodium citrate (0.735 g) and fructose (1.351 g) were dissolved in 100 ml distilled water to prepare HOS solution and it was maintained at 37°C for 5 min before use. Each semen sample (100 μl) was mixed with 1000 μl of HOS solution and incubated at 37°C for 60 min. After incubation, the spermatozoa were fixed with formaldehyde (10% formalin) for subsequent observation of swollen sperm. Such fixation retained the shape of spermatozoa, which could be observed even at a later stage. After placing a drop of incubated well mixed semen sample on a glass slide and covering it with a cover slip, a total of 200 sperms were counted in at least 5 different fields of view and the percentage of sperm that reacted with different swelling patterns of tail were observed.

Assessment of DNA integrity by TUNEL assay 
 
The amount of DNA fragmentation was determined by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay using a commercially available kit (Promega), whereby the free 3-OH ends of DNA are labeled with fluorescein conjugated dUTP by the enzyme terminal deoxynucleotidyl transferase. In order to perform the TUNEL assay, the spermatozoa from the semen sample and those from the post-selection sample were washed with phosphate-buffered saline (PBS) pH 7.2) supplemented with 0.3% albumin and adjusted to a concentration of 20 million/ml; 100 µl of each sample was fixed with 100 µl 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. Once the fixative was removed, it was washed twice with PBS-albumin and permeated with 0.1% Triton X-100 (Sigma) in 0.1% sodium citrate for 2 min in ice bath and was subsequently washed twice with PBS. Before incubation with the TUNEL solution, an additional sample was incubated for 20 min at 37°C with 50 µl (8 U/ µl) of recombinant DNase I (Roche; 10,000 U). All the samples, including the positive control, were incubated in the dark for 1 h at 37°C, with 50 µl of a TUNEL reaction solution (Promega), comprising a deoxyuridine triphosphate (dUTP) solution marked with fluorescein isothiocyanate (FITC) plus the terminal enzyme deoxynucleotidyl transferase (TdT) which enables the specific attachment of uracil to the 3’OH terminal end of a DNA fragment. Samples which had only been incubated with the staining solution (fluorescein-dUTP) without enzyme aggregates were used as negative controls. Once staining was complete, each sample was washed twice with PBS to remove the non-attached solution and resuspended in a final volume of 400 µl and 100 µl of propidium iodide was added at 0.1% in PBS to remove the events with no DNA from the analysis. The samples were read using fluorescence microscopy (Nikon), evaluating at least 100 spermatozoa in duplicate.
 
Statistical analysis
               
The percentage X enrichment by different DGC models are presented as means ± standard error (SE) and data was analyzed by factorial analysis of variance (ANOVA) in a complete randomized design through a statistical software SPSS 20.0 (Statistical Package for the Social Sciences; IBM SPSS Statistics for Windows, Released 2011. Armonk, NY: IBM Corp.) and differences were compared with the post hoc Tukey test at a signifi­cance level of 0.05. The following general linear model was used for the analysis.
   
where
Y  = % x of i^th media, j^th layer and k^th speed.
µ   = Overall mean.
X_i  = Effect of i^th media (i= 1-4).
Y_i  = Effect of j^th layer (j=1-3).
Z_k  = Effect of k^th speed (k=1-2).
(XYZ)_ijk=   Effect of interaction among X, Y and Z.
e_ijk= Random residual.

Percentage of X enrichment by different DGC models                             
 
The percentage enrichment of X bearing sperm in the samples selected by Percoll, Optiprep and Ficoll density gradient centrifugations was found significantly different in comparison to the sucrose density gradient centrifuged semen samples, swimup treated samples and washed semen samples. Enrichment of X bearing sperm by means of discontinuous density gradient centrifugation was found highest for Optiprep DGC model (67.05±3.78) followed by Percoll DGC model (66.32±3.29) and Ficoll DGC model (66.09±4.33), although the difference between  three values was non significant (P<0.05). Previous studies have demonstrated a deviation of upto 90% in favor of females for IVP (invitro production) embryos using Percoll density gradient centrifugation (Blottner et al., 1994). Significant sex ratio deviation to females has also been reported using Percoll, Optiprep and Ficoll discontinuous gradients (Hadi and Timimi, 2013; Lima et al., 2015; Promthep et al., 2016). Enrichment through sucrose DGC model gave the least values i.e., 58.47 ±3.18 (P<0.05) than other DGC models. This may be accounted to the physiochemical properties of sucrose. Sucrose solutions in the concentration range have a high osmolality and are viscous (Kaneshratnam et al., 2012). This might also be the reason behind low viability of sperms enriched through sucrose density gradient models. Percentage of X chromosome content in washed semen samples and semen processed through swim up technique was found 49.63±1.95 and 51.84±1.29 respectively, although the values were not different significantly (P<0.05) with the expected sex ratios of 1:1 from unsorted semen samples.
 
Effect of number of layers and centrifugation speed (g) on percentage X chromosome content enrichment in semen samples
 
Statistical analysis revealed that number of layers and centrifugation speed (g) factors in a density gradient centrifugation model have significant effect on the percentage enrichment of X chromosome content in semen samples (the detail is provided in the supplemental files- Graph S1- S4). Increasing the number of layers of the gradient improved the enrichment of X-bearing sperms. Percentage X bearing sperm enrichment ranged from 61.72±0.281 to 67.312±0 .336 for 3 to 5 layers. Increasing the centrifugation speed from 200xg to 300xg reduced the percentage X enrichment from 66.362±0.257 to 62.696±0.227 respectively. It is observed that separation in density gradient centrifugation, occurs as a result of X and Y bearing sperms density difference. However, when a gradient is centrifuged, the effect of sperm motility is minimized and their mass difference effect is maximized, it makes the heavier sperms reach the bottom faster. Another factor might affect the speed of sperm penetrating through gradient, heavier sperms should settle down faster than lighter sperms; therefore centrifugation speed could positively influence Y bearing sperms moving down the gradient. The lesser the centrifugation speed, the Y bearing sperm (lighter ones) would not reach the bottom towards higher gradient concentration. Increasing the number of layers makes it difficult for Y bearing sperm (lighter ones) to penetrate all the layers and reach the bottom of the gradient. And so, we observed increase in percentage enrichment of X bearing sperm with the increase in number of layers in our study. From our results we can conclude that the enrichment of X bearing sperms was high when we used density gradient model with 5 layer and centrifugation speed of 200xg compared to other density gradient models.
 
Assessment of motility and plasma membrane integrity
 
Motility and plasma membrane integrity are important sperm characteristics for the maintenance of sperm fertility (Singh et al., 2017). Based on the results of percentage X chromosome content enrichment in semen samples by different centrifugation models, model with five layers and centrifugation speed of 200 g for all the density gradient medias were selected viz., P5, O5, F5 and S5 for viability assessment. Motility was found highest for the sperms enriched through model P5 followed by O5, F5 and S5 with the values of 62%, 60%, 52% and 30% respectively. Motility of sperms in washed semen was found to be 70%. Swimup technique processing lowered the motility of sperms to 50%. Plasma membrane integrity was assessed by mean values of HOS positive sperms (Fig 1). More HOS positive cells indicate viability of sperms. Mean value of HOS coiling was found highest for the sperms enriched through model P5 followed by O5, F5 and S5 with the values of 70%, 65%, 58% and 32% respectively. Plasma membrane integrity of sperms in washed semen was found to be 72%. Plasma membrane integrity of sperms processed through swimup technique was found to be 60%.
 

       
We observed reduction in percentage motility and membrane integrity of enriched semen following density gradient centrifugation. This might be due to several factors including osmotic and oxidative stress, as the centrifugation process presents a stress factor to cell organelles and membranes (Malik et al., 2011). Unlike our findings, many studies have reported the significance of density gradient centrifugation to isolate and enrich motile and viable spermatozoa in sperm preparation technique (Malvezzi et al., 2014). But, the reduced values of motility and plasma membrane integrity in our study may be accounted to passage of sperms through five layers of increasing densities, rather than the passage of sperms through only two layers in sperm preparation technique.
 
Assessment of DNA integrity
 
Seminal plasma is an important source of antioxidants. Therefore, separating spermatozoa from seminal plasma during semen processing will result in a prooxidant state. It has been shown that repetitive washing of sperm by serial centrifugation increases ROS production and impairs DNA integrity (Shamshi et al., 2008). Since, sperm DNA integrity is an important parameter of sperm quality in the prognosis of infertility and in the outcome of assisted reproductive procedures (Singh and Agarwal, 2011), we conducted TUNEL assay to assess DNA integrity of spermatozoa enriched through model O6. The TUNEL assay, through the enzymatic incorporation of a marked uracil at the 3’- OH terminal end of a DNA fragment, directly measures damage (Fig 2).
 

 
The percentage of spermatozoa with denatured DNA recovered after density-gradient centrifugation was not found significantly different from that of uncentrifuged semen. These findings suggest that density-gradient centrifugation does not impair DNA integrity. It may be attributed to the well known fact, that chromatin stability is related to the proportion of protamine: histone present in sperm chromatin. The high proportion of protamine: histone present in DNA of bovine sperm can be responsible for the high stability of the chromatin which protects the DNA against the possible damages of the enrichment procedure (Carvalho et al., 2010). Additionally, it is important to indicate that, semen samples with motility of not less than 80% were used for the study and low centrifugation speed i.e 300xg was used for the enrichment procedure.

From our results, Optiprep density gradient model with 5 layers and centrifuged at 200x g appears to be promising  model for enrichment of X spermatozoa from buffalo bull semen based on percentage X enrichment and viability characteristics in terms of motility, HOST and DNA integrity. To conclude, DGC offers a promising approach for production of skewed sex ratio in livestock industry. This can be used as a simple protocol for farmer to reduce the cost and increase the number of female calves in their dairy farm.