Confirmation of positive standards constructed for PLP and SRY genes
The positive standards for PLP and SRY genes were constructed by cloning into a pMD20 linear vector and transformed into DH5α competent cells. A total of 10 randomly picked white colonies were screened for each PLP (out of 46 white colonies) and SRY genes (out of 52 white colonies) by colony PCR and all of them were found to be positive. The plasmid constructs were named as pMD20 T-PLP and pMD20 T-SRY for PLP and SRY gene constructs, respectively.
The PLP and SRY genes were identified on the X- and Y- chromosomes, respectively (Tan and Mahanem, 2015) and were used as gender markers in unsexed and sexed spermatozoa in this study. The PLP fragment was specific for a female that is located on the X- chromosome and was not amplified in Y- Y-chromosome bearing spermatozoa, and vice-versa for the SRY amplification. Both PLP and SRY genes exist as a single copy on X- and Y- chromosomes, respectively in the bovine genome (Tan and Mahanem, 2015). Therefore, every single copy of PLP and SRY sequence detected in qPCR indicated the presence of a respective quantity of X- and Y- chromosome bearing spermatozoa.
Quantification of X and Y- chromosome bearing spermatozoa in unsexed semen
The standard curves were constructed with Ct values on X-axis and copy numbers on Y-axis using pMD20 T-PLP and pMD20 T-SRY positive standards at 10 fold increase in copy numbers ranging from 1.023 x 104 molecules to 1.023 x 1010 molecules. The linear regression curve was set and the equations were obtained as y = 8E + 12e− 0.705x for pMD20 T-PLP and y = 2E + 11e− 0696x for pMD20 T-SRY. The copy number of X and Y-chromosomes for unsexed semen samples of four breeds were analysed by substituting the respective Ct values in the equation that directly corresponds to the number of spermatozoa. The percentage of X and Y- chromosome bearing spermatozoa were estimated; the percentage of X chromosome bearing spermatozoa ranged from 62.88%±0.56 to 71.21±0.93 and Y chromosome bearing spermatozoa ranged from 28.79%±1.26 to 37.12%±1.34 in unsexed semen samples of four breeds (Fig. 1a).
Here, the percentage of PLP- and SRY- genes bearing spermatozoa was determined using SYBR green quantitative real-time PCR in US and XS semen samples. In qPCR analysis, SYBR® Green was chosen as a DNA intercalating fluorescent dye due to several advantages over sequence-specific probes when single plex PCR is performed. The dye is the simplest and least expensive compared to the other known dyes (Leong et al., 2007). However, SYBR® Green dye tends to bind to all double-stranded nucleic acid molecules, hence the accumulation of primer dimers and the amplification of non-specific PCR products can also be detected in SYBR® Green (Lekanne Deprez et al., 2002). To overcome this, a melt curve was run after each cycle to ensure the presence of a single melt peak indicating the absence of nonspecific PCR products. It had been reported that SYBR® Green detection provides a reliable alternative to both the traditional blotting methods and expensive TaqMan protocols (Lee et al., 2006).
The copy numbers below 1 x 104 in qPCR analysis indicated inconsistent CT values as mentioned earlier (Workenhe et al., 2008). Therefore, the copy numbers of the plasmid standards in this study were ranged from 1.0213 x 104-1.0213 x 1010. The PLP and SRY markers exist as single copies in X- and Y- chromosome bearing spermatozoa (Tan and Mahanem, 2015), indicating the copy number directly relates to number of spermatozoa.
In this study the percentage of X chromosome bearing spermatozoa ranged from 62.88%±0.56 to 71.21±0.93 and Y ranged from 28.79%±1.26 to 37.12%±1.34 in US semen samples. However, the studies of Rosenfeld and Roberts (Rosenfeld and Roberts, 2004); Whyte and group (Whyte et al., 2007) revealed 1:1 ratio for X and Y sperms. However, a series of studies conducted on percentage of X and Y bearing chromosome bearing sperms previously suggest that the ratio might differ (Parati et al., 2006; Tan and Mahanem, 2015; Kumari et al., 2019). The deviation from 1:1 ratio might be due to individual variation in sires, number of ejaculations, quantity of X and Y chromosomes in an ejaculation, environmental factors, genetic factors, feeding, nutrition, etc (Brito et al., 2002).
Quantification of X and Y- chromosome bearing spermatozoa in sexed semen
The number of X and Y-chromosome bearing spermatozoa in semen samples of four breeds sexed by flow cytometer were analysed by substituting the respective Ct values in the equation generated in the standard curve (as mentioned in the section above). The percentage of X- chromosome bearing spermatozoa in semen sorted through the flow cytometry technique ranged from 99.02%±3.36 to 99.99%±4.56 whereas Y-chromosome bearing spermatozoa ranged from 0.07–0.98% in the four breeds (Fig. 1b). The semen samples sexed by decapitation of Y-chromosome bearing spermatozoa were available for only three breeds (HF, Jersey and Gir). The percentage of X- chromosome bearing spermatozoa in semen sexed through decapitation of Y-chromosome bearing spermatozoa technique ranged from 95.92%±1.83 to 97.99%±2.74 whereas Y-chromosome bearing spermatozoa ranged from2.1 % to .08 % in the three breeds (Fig. 1c).
The sex-sorted semen straws sorted by either flow cytometry or decapitation of Y chromosome methods following flow cytometry sorting were collected from respective manufacturers. The manufacturers reported more than 90% X-chromosome bearing sperms and less than 10% Y-chromosome bearing sperms for sex-sorted semen sorted by both methods. The quantification has been carried out similar to unsexed semen to quantitatively verify the reports of the manufacturer. Wang and group (Wang et al., 2011) reported the average purity of X sperm in the X enriched samples by flow cytometry was 92% based on qPCR. Welch and Johnson (Welch and Johnson, 1999) reported that the average purity of X sperm in the X enriched samples by flow cytometry was 90% based on qPCR. A previous study (Maleki et al., 2013) reported that there is no significant difference in semen sex ratio in unsorted semen (54.7 ± 0.52% X and 47.6 ± 0.60% Y) and there was a significant difference observed in sorted semen by flow cytometry (93.3 ± 0.08% for X sperms) based on qPCR.
The main challenge we encountered in quantifying X- and Y- chromosome bearing spermatozoa from sex-sorted semen by decapitation of Y chromosome technique is to eliminate the dead Y- chromosome bearing sperms. It was reported that the swim-up technique helps to segregate the live and dead sperms (Magdanz et al., 2019). Briefly, the live sperms swim up by consuming the nutrients in the media which can be collected from the top layer (García-Herreros and Leal, 2014). This method was successfully employed in the current study. The X-chromosome bearing sperms ranged from 95.92–97.99% and Y-chromosome bearing sperms ranged from 2.1–4.08% for sex-sorted semen sorted by decapitation of Y chromosome technique. However, we found no previous data on quantitative analysis of sex-sorted semen sorted by decapitation of Y chromosome technique to compare the findings of the current study. Ours is the first study to report a quantitative evaluation of semen sorted by decapitation of the Y chromosome technique.
The SYBR qPCR technique in the present study is a rapid and reliable technique in quantifying the sex ratio of X- and Y- chromosome bearing spermatozoa in semen samples. This method can be a suitable tool for routine verification of bovine X and Y- chromosome bearing spermatozoa in sorted semen samples or for other related techniques such as quantitative detection of various genes for different applications
Semen quality parameters of the sexed semen
The sperm motility in the US semen samples (65.98% ± 0.23) is relatively better than the XS semen samples (60.88% ± 0.18). The acrosomal integrity of sperms in the US semen samples (75.28% ± 0.05) had relatively fared well than the XS semen samples (70.79% ± 0.26). The percentage of live spermatozoa was also fairly better in US semen samples (67.83% ± 0.42) compared to the XS semen samples (63.58% ± 0.25). The sperm abnormalities were relatively higher in XS semen samples (18.12% ± 0.18) as compared to the US semen samples (13.82% ± 0.02) (Fig. 2).
In the current study, the quality parameters such as sperm motility, acrosomal integrity, live spermatozoa and (fewer) sperm abnormalities for US semen show superior quality over sexed semen. Similar findings were reported previously stating the superiority of unsexed semen over sexed semen in semen quality parameters (Carvalho et al., 2009, 2010; Goncharenko and Pelykh, 2016). It was reported that the semen quality parameters can be influenced by genetic and non-genetic factors (Gopinathan et al., 2018; Bhave et al., 2020). The non-genetic factors include seasonal effects (Nichi et al., 2006; Koivisto et al., 2009; Fiaz et al., 2010), number of ejaculations, temperature, humidity, feeding and nutrition (Isnaini et al., 2019). The genetic factors include individual variation in sires, and the quantity of X and Y chromosomes in an ejaculation (Brito et al., 2002). During sorting of sperm by flow cytometry, sperm passes through nozzle with pressure, dyeing of DNA, passing through an ultraviolet laser beam, electrostatic separation and centrifugation; all of these might lead to alteration of membrane and other changes such as pre capacitation in the sorted sperm leading to decreased semen quality (Kumar et al., 2016).