Table_1_Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction.docx

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.

通过普通牛（Bos taurus）与瘤牛（Bos indicus）杂交获得的杂交公牛，其不育/低育性发生率较高，但相关病因学机制仍未明确。精液评估技术不仅预测可靠性欠佳，且无法与生育力需求维持稳定相关性，因此亟需开发替代方法以实现公牛生育力的精准预测。为此，本研究采用高通量RNA测序（high-throughput RNA sequencing）技术，对高育性与低育性杂交公牛精子的全局差异基因表达情况进行了评估，旨在筛选与杂交公牛生育力相关的转录本（transcripts）。杂交公牛精子中共检测到13563个基因的转录本，其中2093个为高育性公牛特有，5454个为低育性公牛特有。对数据进行标准化处理后，共检测到776个转录本，其中84个为高育性公牛特有，168个为低育性公牛特有。低育性公牛中共有176个转录本表达上调（倍数变化>1），209个转录本表达下调（倍数变化<1）。基因本体（Gene Ontology, GO）分析结果显示，低育性杂交公牛精子中，参与氧化磷酸化通路以及多细胞有机体发育、精子发生、子宫内胚胎发育等生物学过程的转录本呈现下调表达。而低育性公牛特有且上调表达的精子转录本，则主要参与翻译生物学过程及核糖体通路。本研究通过实时荧光定量聚合酶链式反应（RT-qPCR），在12头具有不同生育等级的杂交公牛中对筛选得到的12个精子转录本进行了验证。结果表明，ZNF706、CRISP2、TNP2及TNP1基因的转录丰度在低育性公牛中显著（p<0.05）低于高育性公牛，且与受胎率呈显著正相关（p<0.05）。本研究推断，氧化磷酸化功能受损可能是导致杂交公牛低育性的主要原因，而ZNF706、CRISP2、TNP2及TNP1基因的转录丰度可作为杂交公牛生育力的潜在生物标志物。