Unraveling the Adipose Tissue Proteome of Transition Cows through Severe Negative Energy Balance.

Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum.

Impact of the severity of negative energy balance on gene expression in the subcutaneous adipose tissue of periparturient primiparous Holstein dairy cows: Identification of potential novel metabolic signals for the reproductive system.

The severity of negative energy balance (NEB) in high-producing dairy cows has a high incidence among health diseases. The cow's energy status during early lactation critically affects metabolic and reproductive parameters. The first objective of this study was to investigate by RNA-seq analysis and RT-qPCR the gene expression profile in white adipose tissue and by gene ontology and upstream regulation tools the relationships with energy metabolism and reproduction in two groups of primiparous dairy cows with extreme NEB statuses (NEB < -9 Mcal/day vs. NEB > -9 Mcal/day) around parturition. The second objective was to determine the potential involvement of a new adipokine identified as a candidate for the regulation of ovarian function in our RNA-seq analysis by using bovine primary granulosa culture, thymidine incorporation to determine cell proliferation and ELISA assays to measure progesterone secretion. The RNA-seq analysis revealed that 514 genes were over-expressed and 695 were under-expressed in the adipose tissue of cows with severe NEB (SNEB) and cows with moderate NEB (MNEB) during the -4 and 16 wkpp period. In addition, 491 genes were over-expressed and 705 genes were under-expressed in the adipose tissue of SNEB cows compared to MNEB cows. Among these differently expressed genes (DEGs), 298 were related to metabolic functions and 264 to reproductive traits. A set of 19 DEGs were validated by RT-qPCR, including CCL21 (C-C motif chemokine ligand 21). Moreover, CCL21, a gene known to be secreted by adipose tissue, was chosen for further analysis in plasma and ovaries. The use of next-generation sequencing technologies allowed us to characterise the transcriptome of white adipose tissue from primiparous cows with different levels of NEB during lactation. This study highlighted the alteration of the expression of genes related to lipid metabolism, including CCL21, which is released in the bloodstream and associated with the in vitro regulation of ovarian functions.

Possible involvement of the RARRES2/CMKLR1-system in metabolic and reproductive parameters in Holstein dairy cows.

BACKGROUND: In dairy cows, the energy cost of milk yield results in a negative energy balance (EB) and body fat mobilization that impairs reproductive efficiency. Emerging evidence suggests that the novel adipokines, Retinoic acid receptor responder protein 2 (RARRES2), and its main receptor, Chemokine-like receptor 1 (CMKLR1) are involved in the regulation of metabolic and ovarian functions. So, we investigated in a first experiment the plasma RARRES2, and RARRES2 and CMKLR1 mRNA expression levels in subcutaneous adipose tissue (SAT) and granulosa cells (GC) at different times of body fat mobilization in dairy cows (4, 8, 20 and 44 weeks postpartum, wk. pp. for SAT and 8, 20 and 44 wk. pp. for GC). Then, in a second experiment we examined the effect of high (HE) and low energy (LE) diets on the RARRES2 system and its links with metabolic and reproductive parameters. METHODS: The first experiment included 9 animals fed with HE diet from 4 to 44 wk. pp. and the second one included animals fed either a HE diet (n = 8) or a LE diet (n = 8) from - 4 to 16 wk. peripartum. In both experiments, various metabolic and reproductive parameters were determined and associated with plasma RARRES2 as measured by bovine ELISA. RARRES2 and CMKLR1 mRNA expression levels were analyzed by RT-qPCR in SAT after biopsy and GC after aspiration of follicles. RESULTS: Plasma RARRES2 levels were higher at 4 wk. pp. as compared to 20 and 44 wk. pp. and they were positively correlated with body fat mobilization and milk yield. RARRES2 and CMKLR1 mRNA expression levels increased from 4 to 8 wk. pp. (fat mobilization, EB < 0) and remained unchanged at 20 and 44 wk. pp. (fat reconstitution, EB > 0) as compared to 4 wk. pp. in SAT. RARRES2 and CMKLR1 mRNA levels decreased from 8 to 44 wk. pp. in GC from small follicles. In the second experiment, plasma RARRES2 increased from - 4 to 8 wk. peripartum similarly in both LE and HE cows. In addition, the area under of plasma RARRES2 curve was highly negatively associated with the number of small follicles obtained in HE animals during the cycle before the first artificial insemination. In SAT of HE cows, RARRES2 mRNA expression decreased at 1 wk. pp. compared to - 4 and 16 wk. peripartum whereas opposite expression patterns were obtained for CMKLR1. Similar results were observed for CMKLR1 mRNA expression in LE cows while there was no variation in RARRES2 mRNA expression. Moreover, RARRES2 mRNA was higher expressed in LE than in HE cows at 1 wk. pp. CONCLUSIONS: The lactation-induced fat and energy mobilization influenced plasma RARRES2 profile and mRNA expression pattern of RARRES2 and CMKLR1 similarly in both SAT and GC. In addition, the energy content of the diet did not affect plasma RARRES2 but it altered RARRES2 mRNA expression in SAT and the area under the curve of plasma RARRES2 that was negatively associated to the number of small follicles in HE animals. Thus, RARRES2 could be a metabolic or ovarian signal involved in the interactions between metabolic and reproductive functions in dairy cows.

Effect of a long-chain n-3 polyunsaturated fatty acid-enriched diet on adipose tissue lipid profiles and gene expression in Holstein dairy cows.

The objective of this study was to determine whether fish oil supplement has an effect on adipose tissue lipid profiles and gene expression in postpartum dairy cows. Holstein cows were supplemented with either long-chain n-3 polyunsaturated fatty acid (PUFA; protected fish oil) or control PUFA (n-6; toasted soybeans) for 2mo after calving (n=23 per diet). These cows showed no difference in milk production or metabolic parameters, but exhibited a tendency toward a decrease in early embryo mortality rate after artificial insemination. We hypothesized that, in addition to this effect, modifications in adipose tissue (AT) gene expression and lipid profiles would occur in response to diet. Subcutaneous AT samples were thus collected from the dewlaps of n-3 and n-6 dairy cows at 1mo antepartum, and 1wk, 2mo, and 5mo postpartum for the analysis of lipids and gene expression. Lipid profiles were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in both positive and negative modes. We found 37 lipid species in the 200 to 1,200 m/z range, which differed between the n-3 and control groups, suggesting that the n-3 supplement affected the lipid composition through the enrichment of lipids integrating long-chain PUFA from fish oil sources: eicosapentaenoic and docosahexaenoic acid. Moreover, a decrease in triacylglycerolipids was observed in AT of n-3 supplemented cows. The expression of 44 genes involved in fatty acid metabolism and the adipokine system was assessed by real-time reverse-transcription PCR. Hierarchical clustering, according to either postpartum stage or diet, enabled us to group genes exhibiting similar kinetic properties during lactation or by those that varied in similar ways after n-3 supplementation, respectively. Among the genes exhibiting a dietary effect, FABP4, LIPE, CD36, and PLIN1 were overexpressed in n-3 AT samples compared with the control, suggesting an increase in lipolysis due to n-3 supplementation, which was reflected on lipolytic activity at the protein level (i.e., protein expression of fatty acid binding protein 4, phosphorylated perilipin 1, and phosphorylated hormone-sensitive lipase). This increase in lipolysis is relevant to the decrease in triglycerides observed in these samples. Gene expression analyses between n-3 and control AT samples also suggested that the n-3 diet could modulate the secretory functions of AT, possibly by affecting adipokine expression; however, this has to be confirmed at the protein level.

Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.

The objective of this study was to determine the effect of a rumen-protected fish oil supplement on the production and reproduction variables in postpartum dairy cows. Holstein cows (n=46) were given a basal total mixed diet plus one PUFA supplement: n-3 (n-3; protected fish oil; 1% dry matter intake (DMI); n=23) or control (n-6; toasted soybeans; 1.8% DMI; n=23), in a switchback design over two consecutive lactations. Supplements were added to the diet between calving and 2 months after calving to assess the effect on growth and maturation of ovarian follicles from which ovulation occurred around the day of insemination. Body weight (BW), milk yield (MY) and composition, dry matter intake (DMI), energy balance (EB), subcutaneous fat thickness, plasma fatty acid composition, plasma nonesterified fatty acids (NEFA), glucose and urea concentrations, follicular activity, embryo mortalities and fertility (conception rate after first AI, AI1) were assessed. BW, MY, DMI, plasma NEFA, glucose and urea were unaffected by the diet. There was a trend of an increased number of large follicles (diameter≥10mm) with the n-3 dietary supplementation (P=0.06) and a decrease in infertility or early embryo mortality rate 21 days after AI, 13.5% in the n-3 compared with 38.8% in the n-6 group (P=0.09), with no effect on the conception rate at 35d or 90d after AI1. These data suggest that the effect seen on ovarian variables is not associated with an effect on production and metabolic variables and is specific to n-3 PUFA supplementation. Further studies are necessary to determine whether DHA or EPA enhances fertility in lactating dairy cattle.

Resistin in dairy cows: plasma concentrations during early lactation, expression and potential role in adipose tissue.

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRß, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

Expression of adipokine and lipid metabolism genes in adipose tissue of dairy cows differing in a female fertility quantitative trait locus.

We have previously characterized 2 haplotypes (Fertil+ and Fertil-) of Holstein dairy cows differing in 1 female fertility quantitative trait locus (QTL) located on chromosome 3 (QTL-Fert-F-BTA3) between positions 9.8 and 13.5 cM. This QTL is composed of 124 genes, some of them being involved in metabolism or reproduction. Primiparous Fertil+ and Fertil- cows exhibited 69 and 39% pregnancy rate at first service, respectively. A difference in plasma nonesterified fatty acid concentrations observed between both haplotypes might indicate a difference in adipose tissue mobilization. We compared adipose tissue gene expression in Fertil+ and Fertil- cows during their second lactation, at 2 physiological stages, implying either intense lipid mobilization (1 wk postpartum) or fat storage (5 mo of gestation). We investigated by reverse-transcription quantitative PCR the mRNA gene expression of 5 positional candidate genes located in the QTL-Fert-F-BTA3, as well as 18 other functional candidate genes encoding proteins involved in lipid metabolism and several adipokines. Among them, genes involved in either lipolysis or lipogenesis were chosen as controls because they were previously described in dairy cow adipose tissue. A hierarchical clustering was performed to group genes according to their expression pattern, allowing 2 clusters to be determined. Cluster 1 was composed of genes that were overexpressed during mobilization (ADIPOQ, ADIPOR2, LIPE, FABP4, PLIN1, RARRES, LEPR, and CPT1A) and cluster 2 of genes overexpressed during reconstitution of body reserves (ACACA, FASN, and SCD). Genes belonging to cluster 1 (LIPE, FABP4, PLIN1, and CPT1A) are known to be involved in lipolysis and fatty acid oxidation, and genes belonging to cluster 2 (ACACA, FASN, and SCD) are known to be involved in fatty acid synthesis. The expression of 5 genes from cluster 1 was correlated to plasma nonesterified fatty acid levels and thus to mobilization of body reserves in dairy cows (ADIPOQ, ADIPOR2, LIPE, PLIN1, and FABP4). During the mobilization stage, none of the positional candidate genes belonging to QTL-Fert-F-BTA3 (ADAR, MTX1, SHC1, SPTA1, and PAQR6) showed a difference in expression between the 2 haplotypes. Interestingly, ADIPOQ and ADIPOR2 were the only genes showing a significant mRNA overexpression in Fertil- cows at the mobilization stage. Further studies focusing on plasma adiponectin level and adipokine actions on the ovary are needed to investigate its potential role in dairy cow fertility.

Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow.

The major limitation to the development of embryo production in cattle is the strong between-animal variability in ovulatory response to FSH-induced superovulation, mainly due to differences in ovarian activity at the time of treatment. This study aimed to establish whether anti-Müllerian hormone (AMH) was an endocrine marker of follicular populations in the cow, as in human, and a possible predictor of the ovarian response to superovulation. Anti-Müllerian hormone concentrations in plasma varied 10-fold between cows before treatment and were found to be highly correlated with the numbers of 3- to 7-mm antral follicles detected by ovarian ultrasonography before treatment (r=0.79, P<0.001) and the numbers of ovulations after treatment (r=0.64, P<0.01). Between-animal differences in AMH concentrations were found to be unchanged after a 3-mo delay (r=0.87, P<0.01), indicating that AMH endocrine levels were characteristic of each animal on a long-term period. The population of healthy 3- to 7-mm follicles was the main target of superovulatory treatments, contained the highest AMH concentrations and AMH mRNA levels compared with larger follicles, and contributed importantly to AMH endocrine levels. In conclusion, AMH was found to be a reliable endocrine marker of the population of small antral gonadotropin-responsive follicles in the cow. Moreover, AMH concentrations in the plasma of individuals were indicative of their ability to respond to superovulatory treatments.