Transcriptome profiles in peripheral white blood cells at the time of artificial insemination discriminate beef heifers with different fertility potential. Bos taurus

Background: Infertility is a longstanding limitation in livestock production with important economic impact for the cattle industry. Female reproductive traits are polygenic and lowly heritable in nature, thus selection for fertility is challenging. Beef cattle operations leverage estrous synchronization in combination with artificial insemination (AI) to breed heifers and benefit from an early and uniform calving season. A couple of weeks following AI, heifers are exposed to bulls for an opportunity to become pregnant by natural breeding (NB), but they may also not become pregnant during this time period. Focusing on beef heifers, in their first breeding season, we hypothesized that: a- at the time of AI, the transcriptome of peripheral white blood cells (PWBC) differs between heifers that become pregnant to AI and heifers that become pregnant late in the breeding season by NB or do not become pregnant during the breeding season; and b- the ratio of transcript abundance between genes in PWBC classifies heifers according to pregnancy by AI, NB, or failure to become pregnant. Results: We generated RNA-sequencing data from 23 heifers from two locations (A: six AI-pregnant and five NB-pregnant; and B: six AI-pregnant and six non-pregnant). After filtering out lowly expressed genes, we quantified transcript abundance for 12,538 genes. The comparison of gene expression levels between AI-pregnant and NB-pregnant heifers yielded 18 differentially expressed genes (DEGs) (ADAM20, ALDH5A1, ANG, BOLA-DQB, DMBT1, FCER1A, GSTM3, KIR3DL1, LOC107131247, LOC618633, LYZ, MNS1, P2RY12, PPP1R1B, SIGLEC14, TPPP, TTLL1, UGT8, eFDR≤0.02). The comparison of gene expression levels between AI-pregnant and non-pregnant heifers yielded six DEGs (ALAS2, CNKSR3, LOC522763, SAXO2, TAC3, TFF2, eFDR≤0.05). We calculated the ratio of expression levels between all gene pairs and assessed their potential to classify samples according to experimental groups. Considering all samples, relative expression from two gene pairs correctly classified 10 out of 12 AI-pregnant heifers (P=0.0028) separately from the other 11 heifers (NB-pregnant, or non-pregnant). Conclusion: The transcriptome profile in PWBC, at the time of AI, is associated with the fertility potential of beef heifers. Transcript levels of specific genes may be further explored as potential classifiers, and thus selection tools, of heifer fertility. Overall design: Peripheral white blood cells (PWBC) were collected from heifers at the time of artificial insemination. We generated transcriptome data from PWBC and contrasted the gene expression values between heifers that became pregnant and did not became pregnant.

背景：不孕症是家畜生产中长期存在的瓶颈，对养牛业具有重大经济影响。雌性繁殖性状本质上属于多基因遗传且遗传力较低，因此针对生育能力的选育工作颇具挑战。肉牛养殖企业常通过发情同步化结合人工授精（artificial insemination, AI）对青年母牛进行配种，以实现产犊季早且整齐的目标。在人工授精后的数周内，会将青年母牛与公牛同栏，使其有机会通过自然配种（natural breeding, NB）受孕，但部分个体在此期间仍无法受孕。本研究以首个配种季的肉牛青年母牛为对象，提出如下假设：其一，在人工授精时，通过人工授精受孕的青年母牛与在配种季后期通过自然配种受孕或整个配种季均未受孕的青年母牛，其外周血白细胞（peripheral white blood cells, PWBC）的转录组存在差异；其二，外周血白细胞中基因的转录本丰度可用于区分青年母牛的受孕类型：人工授精受孕、自然配种受孕或未受孕。
结果：本研究从两个试验地点共收集23头青年母牛的样本并进行RNA测序：地点A包含6头人工授精受孕个体与5头自然配种受孕个体；地点B包含6头人工授精受孕个体与6头未受孕个体。过滤低表达基因后，我们共定量得到12538个基因的转录本丰度。对比人工授精受孕与自然配种受孕青年母牛的基因表达水平，共得到18个差异表达基因（differentially expressed genes, DEGs）：ADAM20、ALDH5A1、ANG、BOLA-DQB、DMBT1、FCER1A、GSTM3、KIR3DL1、LOC107131247、LOC618633、LYZ、MNS1、P2RY12、PPP1R1B、SIGLEC14、TPPP、TTLL1、UGT8，校正后错误发现率（eFDR）≤0.02。对比人工授精受孕与未受孕青年母牛的基因表达水平，共得到6个差异表达基因（DEGs）：ALAS2、CNKSR3、LOC522763、SAXO2、TAC3、TFF2，eFDR≤0.05。我们计算了所有基因对的表达水平比值，并评估其对样本进行试验分组分类的潜力。综合所有样本来看，基于2个基因对的相对表达量可将12头人工授精受孕青年母牛中的10头正确区分出来（P=0.0028），与其余11头（自然配种受孕或未受孕）个体区分开。
结论：人工授精时外周血白细胞的转录组特征与肉牛青年母牛的生育潜力相关。特定基因的转录水平可进一步作为青年母牛生育能力的分类标志物乃至选育工具进行深入研究。
整体试验设计：于人工授精时采集青年母牛的外周血白细胞，对其进行转录组测序，并对比受孕与未受孕青年母牛的基因表达水平。