Exome sequencing identifies variants associated with semen quality in Holstein Friesian and Hallikar bulls

Effective fertility of bulls is dependent on semen quality, often determined based on standard semen evaluation tests. Here we report Whole Exome Sequencing (WES) of 12 bulls from two breeds Holstien Friesian and Hallikar selected based on Ejaculate Rejection Rate (ERR). We explored the possibility of identifying genetic variants from the conserved protein coding regions of genome. A total of 10,510 SNPs and 10.236 INDELs were identified post alignment against reference genome (ARS-UCD 1.2) and were annotated using SnpEff. The number of variants with high and modifier functional impact detected were 145 and 19,122, respectively. Genetic variants common to both high and low ERR group bulls among Holstein Friesian were 08 and in Hallikarthe common variants were 51. Prominent genesviz. UCP2, PANK2, GPD2, PTPRG, LARP7, EZH1, DENND1B and TDRD9 with a role in determining the semen quality were observed to be carriers of the genetic variant.


Introduction
Improving the efficiency of reproductive ability of dairy cattle has been the biggest challenge for the dairy industry [1]. Selecting sires for improved fertility has been a task owing to its low heritability, compared to that of production traits [2-3] in cattle. While much attention had have been diverted on improving cow fertility traits, there requires a committed focus in understanding bull fertility traits [1].The fertility of the breeding population either sire or dam determines the economic viability of livestock husbandry. More precisely sire fertility has greater impact than of the dam's, as it is understood that sires are more than half the herd. Over the past few decades cattle breeds have been reared for improved milk production and beef, this preferential selection has resulted in development of breedswith specialised traits. The intensified preferential selection strategy has resulted in desirable production output, but with a consequent reduction in fertility traits owing to an antagonistic relationship between milk yield and fertility [4]. In the present circumstances focus should be on the need to upgrade the selection methodology with an aimto improve the fertility rate and produce viable off-springs to achieve intended target. This could probably be realised by including genetic information underlying traits such as semen functional and quality traits into selection strategy. Both candidate gene and genome-wide approaches have been successfully employed to study the genomic architecture of bull and have resulted in identifying bio-markers defining fertility [1]. A gene panel approach through genes associated with fertility and pathways regulating it, may be used as a filter to identify novel genetic variants. Hence, this study was planned with an aim to utilize whole exome sequencing, to unravel possible variants determining the bull semen quality in Hallikar a native cattle breed of Karnataka, India, known for its high endurance, hardiness and Holstein Friesian known for its high milk producing ability.

Materials and Method
Holstein Friesian bulls maintained at Nandini Sperm Station, Hesarghatta, Bengaluru Karnataka, India and Hallikar bulls maintained at State Semen Collection Centre, Hesaraghatta, Bengaluru, Karnataka, India were used for the present study. Records on semen quality obtained for a period of one year from August 2019 to August 2020 were utilized to calculate Ejaculate Rejection Rate (ERR) using the formula: ERR (%) = Number of ejaculates found unsuitable for freezing ×100/ Total number of ejaculates obtained from the bull [5].
Based on ERR six (06) bulls each from Holstein Friesian and Hallikar breeds were selected and grouped as three (03) with lower ERR and three (03) with higher ERR (S 1 ). About 4 ml of blood from each selected bull were collected aseptically in a vacutainer tube containing 0.5% EDTA and gDNA was isolated using the Modified High Salt Extraction method. gDNA samples with 260/280 ratio ≥ 1.8 were utilised for the subsequent sequencing. Exome libraries were prepared by using Agilent SureSelect Bovine All Exon panel™ (kit) following the manufacturer's protocol.
Sequencing and Bio-informatic Analysis.
Paired-end sequencing was carried out on IlluminaNovaseq 6000 platform, with a sequencing parameter of 150×2 at 100X depth. Data obtained through high throughput sequencing was subjected for a quality check using FastQC v0.11.9

Conclusion:
The present study has resulted in identifying genetic variants in the conserved protein-coding regions of the genome. The variants identified were observed to be part of genes regulating major semen functional traits. Thus emphasising that Whole Exome Sequencing could be a viable option to identify genetic variants determining traits pertaining to reproduction and can be used as a tool in selecting bulls for breeding strategies.