• Effect of dietary restriction and subsequent re-alimentation on the transcriptional profile of hepatic tissue in cattle

      Keogh, Kate; Kenny, David A; Cormican, Paul; Kelly, Alan K; Waters, Sinead M (Biomed Central, 2016-03-17)
      Background Compensatory growth (CG) is an accelerated growth phenomenon observed in animals upon re-alimentation following a period of dietary restriction. It is typically utilised in livestock systems to reduce feed costs during periods of reduced feed availability. The biochemical mechanisms controlling this phenomenon, however, are yet to be elucidated. This study aimed to uncover the molecular mechanisms regulating the hepatic expression of CG in cattle, utilising RNAseq. RNAseq was performed on hepatic tissue of bulls following 125 days of dietary restriction (RES) and again following 55 days of subsequent re-alimentation during which the animals exhibited significant CG. The data were compared with those of control animals offered the same diet on an ad libitum basis throughout (ADLIB). Elucidation of the molecular control of CG may yield critical information on genes and pathways which could be targeted as putative molecular biomarkers for the selection of animals with improved CG potential. Results Following a period of differential feeding, body-weight and liver weight were 161 and 4 kg higher, respectively, for ADLIB compared with RES animals. At this time RNAseq analysis of liver tissue revealed 1352 significantly differentially expressed genes (DEG) between the two treatments. DEGs indicated down-regulation of processes including nutrient transport, cell division and proliferation in RES. In addition, protein synthesis genes were up-regulated in RES following a period of restricted feeding. The subsequent 55 days of ad libitum feeding for both groups resulted in the body-weight difference reduced to 84 kg, with no difference in liver weight between treatment groups. At the end of 55 days of unrestricted feeding, 49 genes were differentially expressed between animals undergoing CG and their continuously fed counterparts. In particular, hepatic expression of cell proliferation and growth genes were greater in animals undergoing CG. Conclusions Greater expression of cell cycle and cell proliferation genes during CG was associated with a 100 % recovery of liver weight during re-alimentation. Additionally, an apparent up-regulation in capacity for cellular protein synthesis during restricted feeding may contribute to and sustain CG during re-alimentation. DEGs identified are potential candidate genes for the identification of biomarkers for CG, which may be incorporated into future breeding programmes.
    • Effect of genotype on duodenal expression of nutrient transporter genes in dairy cows

      Waters, Sinead M.; Keogh, Kate; Buckley, Frank; Kenny, David A (Biomed Central, 2013-12-09)
      Background Studies have shown clear differences between dairy breeds in their feed intake and production efficiencies. The duodenum is critical in the coordination of digestion and absorption of nutrients. This study examined gene transcript abundance of important classes of nutrient transporters in the duodenum of non lactating dairy cows of different feed efficiency potential, namely Holstein-Friesian (HF), Jersey (JE) and their F1 hybrid. Duodenal epithelial tissue was collected at slaughter and stored at -80°C. Total RNA was extracted from tissue and reverse transcribed to generate cDNA. Gene expression of the following transporters, namely nucleoside; amino acid; sugar; mineral; and lipid transporters was measured using quantitative real-time RT-PCR. Data were statistically analysed using mixed models ANOVA in SAS. Orthogonal contrasts were used to test for potential heterotic effects and spearman correlation coefficients calculated to determine potential associations amongst gene expression values and production efficiency variables. Results While there were no direct effects of genotype on expression values for any of the genes examined, there was evidence for a heterotic effect (P < 0.05) on ABCG8, in the form of increased expression in the F1 genotype compared to either of the two parent breeds. Additionally, a tendency for increased expression of the amino acid transporters, SLC3A1 (P = 0.072), SLC3A2 (P = 0.081) and SLC6A14 (P = 0.072) was also evident in the F1 genotype. A negative (P < 0.05) association was identified between the expression of the glucose transporter gene SLC5A1 and total lactational milk solids yield, corrected for body weight. Positive correlations (P < 0.05) were also observed between the expression values of genes involved in common transporter roles. Conclusion This study suggests that differences in the expression of sterol and amino acid transporters in the duodenum could contribute towards the documented differences in feed efficiency between HF, JE and their F1 hybrid. Furthermore, positive associations between the expression of genes involved in common transporter roles suggest that these may be co-regulated. The study identifies potential candidates for investigation of genetic variants regulating nutrient transport and absorption in the duodenum in dairy cows, which may be incorporated into future breeding programmes.
    • Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology

      Matthews, Daragh; Diskin, Michael G; Kenny, David A; Creevey, Christopher J; Keogh, Kate; Waters, Sinead M (Biomed Central, 2017-11-09)
      Background Negative energy balance (NEB) is an imbalance between energy intake and energy requirements for lactation and body maintenance affecting high-yielding dairy cows and is of considerable economic importance due to its negative impact on fertility and health in dairy herds. It is anticipated that the cow hypothalamus experiences extensive biochemical changes during the early post partum period in an effort to re-establish metabolic homeostasis. However, there is variation in the tolerance to NEB between individual cows. In order to understand the genomic regulation of ovulation in hypothalamic tissue during NEB, mRNA transcriptional patterns between tolerant and sensitive animals were examined. A short term dietary restriction heifer model was developed which induced abrupt onset of anoestrus in some animals (Restricted Anovulatory; RA) while others maintained oestrous cyclicity (Restricted Ovulatory; RO). A third control group (C) received a higher level of normal feeding. Results A total of 15,295 genes were expressed in hypothalamic tissue. Between RA and C groups 137 genes were differentially expressed, whereas between RO and C, 32 genes were differentially expressed. Differentially expressed genes were involved in the immune response and cellular motility in RA and RO groups, respectively, compared to C group. The largest difference between groups was observed in the comparison between RA and RO heifers, with 1094 genes shown to be significantly differentially expressed (SDE). Pathway analysis showed that these SDE genes were associated with 6 canonical pathways (P < 0.01), of which neuroactive ligand-receptor interaction was the most significant. Within the comparisons the main over-represented pathway functions were immune response including neuroprotection (CXCL10, Q1KLR3, IFIH1, IL1 and IL8; RA v C and RA v RO); energy homeostasis (AgRP and NPY; RA v RO); cell motility (CADH1, DSP and TSP4; RO v C) and prevention of GnRH release (NTSR1 IL1α, IL1β, NPY and PACA; RA v RO). Conclusions This information will assist in understanding the genomic factors regulating the influence of diet restriction on fertility and may assist in optimising nutritional and management systems for the improvement in reproductive performance.
    • Examination of the molecular control of ruminal epithelial function in response to dietary restriction and subsequent compensatory growth in cattle

      O’Shea, Emma; Waters, Sinéad M; Keogh, Kate; Kelly, Alan K; Kenny, David A (2016-09-15)
      Abstract Background The objective of this study was to investigate the effect of dietary restriction and subsequent compensatory growth on the relative expression of genes involved in volatile fatty acid transport, metabolism and cell proliferation in ruminal epithelial tissue of beef cattle. Sixty Holstein Friesian bulls (mean liveweight 370 ± 35 kg; mean age 479 ± 15 d) were assigned to one of two groups: (i) restricted feed allowance (RES; n = 30) for 125 d (Period 1) followed by ad libitum access to feed for 55 d (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). Target growth rate for RES was 0.6 kg/d during Period 1. At the end of each dietary period, 15 animals from each treatment group were slaughtered and ruminal epithelial tissue and liquid digesta harvested from the ventral sac of the rumen. Real-time qPCR was used to quantify mRNA transcripts of 26 genes associated with ruminal epithelial function. Volatile fatty acid analysis of rumen fluid from individual animals was conducted using gas chromatography. Results Diet × period interactions were evident for genes involved in ketogenesis (BDH2, P = 0.017), pyruvate metabolism (LDHa, P = 0.048; PDHA1, P = 0.015) and cellular transport and structure (DSG1, P = 0.019; CACT, P = 0.027). Ruminal concentrations of propionic acid (P = 0.018) and n-valeric acid (P = 0.029) were lower in RES animals, compared with ADLIB, throughout the experiment. There was also a strong tendency (P = 0.064) toward a diet × period interaction for n-butyric with higher concentrations in RES animals, compared with ADLIB, during Period 1. Conclusions These data suggest that following nutrient restriction, the structural integrity of the rumen wall is compromised and there is upregulation of genes involved in the production of ketone bodies and breakdown of pyruvate for cellular energy. These results provide an insight into the potential molecular mechanisms regulating ruminal epithelial absorptive metabolism and growth following nutrient restriction and subsequent compensatory growth.
    • Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue

      Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David A; Waters, Sinead M; McGee, Mark; Kelly, Alan K; McCabe, Matthew S (Biomed Central, 2017-05-02)
      Background Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Results Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences. Conclusions The microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.