Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction

Abstract

Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.

Cellular and Molecular Insights Into the Etiology of Subfertility/Infertility in Crossbred Bulls (Bos taurus × Bos indicus): A Review

“…Post-chilled sperm characteristics of crossbred spermatozoa by next generation RNA Sequencing revealed the transcripts for 13,814 genes, which are highly related to ribosome, spliceosome and oxidative phosphorylation pathways (Prakash et al, 2020(Prakash et al, , 2021, whereas microarray analysis revealed the expression of 19,454 genes in Vrindavani crossbred bull sperm (Yathish et al, 2017). The PRM1 is an abundant transcript found in spermatozoa of crossbred bulls (Singh et al, 2019;Prakash et al, 2020), which is also abundant in Bos indicus (Raval et al, 2019) and Bos taurus (Card et al, 2013;Selvaraju et al, 2017) bulls.…”

“…When two different species or breeds are crossed to produce hybrid offspring, the compatibility between spermatozoon and oocyte is altered ( Jodar et al, 2013 ); this compatibility is essential for the transfer of RNA-based information to a chromatized state (Consolidation process). Global transcriptomic profiling of crossbred spermatozoa by next generation RNA Sequencing revealed the transcripts for 13,814 genes, which are highly related to ribosome, spliceosome and oxidative phosphorylation pathways ( Prakash et al, 2020 , 2021 ), whereas microarray analysis revealed the expression of 19,454 genes in Vrindavani crossbred bull sperm ( Yathish et al, 2017 ). The PRM1 is an abundant transcript found in spermatozoa of crossbred bulls ( Singh et al, 2019 ; Prakash et al, 2020 ), which is also abundant in Bos indicus ( Raval et al, 2019 ) and Bos taurus ( Card et al, 2013 ; Selvaraju et al, 2017 ) bulls.…”

“…Several studies used genomics, transcriptomics, proteomics and metabolomics approach to ascertain molecular determinants of bull fertility. Few studies used high throughput techniques for assessment of transcriptomic, proteomic and metabolomic differences between semen of high- and low-fertile bulls and identified potential molecules for fertility prediction ( Peddinti et al, 2008 ; D’Amours et al, 2010 ; Card et al, 2013 , 2017 ; Prakash et al, 2021 ). While these approaches offer a great scope for prediction of bull fertility, reported variations in sperm molecules among different breeds ( Aslam et al, 2015 ) indicate that fertility associated semen molecules might vary with breed, which in turn demand identification of breed specific fertility markers.…”

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock

“…Thus, they may have an association with potentiated DNAs for gene expression ( Jodar et al, 2013 ). Further, the sperm transcriptome profiling using Microarray ( Das et al, 2013 ; Zhang Y. et al, 2017 ), and RNA-seq ( Gòdia et al, 2018 ) have identified a suite of RNAs in spermatozoa ( Sendler et al, 2013 ) that included both coding and non-coding transcripts and were associated with regulation of various biological functions such as chromatin repackaging, genomic imprinting, early embryonic development ( Das et al, 2013 ), and post-fertilization events ( Prakash et al, 2021 ). The spRNA profile also differed between low-motile and high-motile spermatozoa ( Lambard et al, 2004 ) and between fertile and infertile men ( Wang et al, 2004 ; Ostermeier et al, 2005 ).…”

“…Altered miRNAs expression has also been found in the testis with non-obstructive azoospermia ( Lian et al, 2009 ). Thus, the expression level of spRNAs and seminal RNAs may have potential use in studying the post-spermatogenesis events and detecting defects in sperm function ( Prakash et al, 2021 ). A layout of whole transcriptome studies in major livestock species is presented in Figure 1 .…”