Feed efficiency in dairy sheep: An insight from the milk transcriptome.

Introduction: As higher feed efficiency in dairy ruminants means a higher capability to transform feed nutrients into milk and milk components, differences in feed efficiency are expected to be partly linked to changes in the physiology of the mammary glands. Therefore, this study aimed to determine the biological functions and key regulatory genes associated with feed efficiency in dairy sheep using the milk somatic cell transcriptome. Material and methods: RNA-Seq data from high (H-FE, n = 8) and low (L-FE, n = 8) feed efficiency ewes were compared through differential expression analysis (DEA) and sparse Partial Least Square-Discriminant analysis (sPLS-DA). Results: In the DEA, 79 genes were identified as differentially expressed between both conditions, while the sPLS-DA identified 261 predictive genes [variable importance in projection (VIP) > 2] that discriminated H-FE and L-FE sheep. Discussion: The DEA between sheep with divergent feed efficiency allowed the identification of genes associated with the immune system and stress in L-FE animals. In addition, the sPLS-DA approach revealed the importance of genes involved in cell division (e.g., KIF4A and PRC1) and cellular lipid metabolic process (e.g., LPL, SCD, GPAM, and ACOX3) for the H-FE sheep in the lactating mammary gland transcriptome. A set of discriminant genes, commonly identified by the two statistical approaches, was also detected, including some involved in cell proliferation (e.g., SESN2, KIF20A, or TOP2A) or encoding heat-shock proteins (HSPB1). These results provide novel insights into the biological basis of feed efficiency in dairy sheep, highlighting the informative potential of the mammary gland transcriptome as a target tissue and revealing the usefulness of combining univariate and multivariate analysis approaches to elucidate the molecular mechanisms controlling complex traits.

Plasma and milk metabolomics in lactating sheep divergent for feed efficiency.

Enhancing the ability of animals to convert feed into meat or milk by optimizing feed efficiency (FE) has become a priority in livestock research. Although untargeted metabolomics is increasingly used in this field and may improve our understanding of FE, no information in this regard is available in dairy ewes. This study was conducted to (1) discriminate sheep divergent for FE and (2) provide insights into the physiological mechanisms contributing to FE through high-throughput metabolomics. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) technique was applied to easily accessible animal fluids (plasma and milk) to assess whether their metabolome differs between high- and low-feed efficient lactating ewes (H-FE and L-FE groups, respectively; 8 animals/group). Blood and milk samples were collected on the last day of the 3-wk period used for FE estimation. A total of 793 features were detected in plasma and 334 in milk, with 100 and 38 of them, respectively, showing differences between H-FE and L-FE. The partial least-squares discriminant analysis separated both groups of animals regardless of the type of sample. Plasma allowed the detection of a greater number of differential features; however, results also supported the usefulness of milk, more easily accessible, to discriminate dairy sheep divergent for FE. Regarding pathway analysis, nitrogen metabolism (either anabolism or catabolism) seemed to play a central role in FE, with plasma and milk consistently indicating a great impact of AA metabolism. A potential influence of pathways related to energy/lipid metabolism on FE was also observed. The variable importance in the projection plot revealed 15 differential features in each matrix that contributed the most for the separation in H-FE and L-FE, such as l-proline and phosphatidylcholine 20:4e in plasma or l-pipecolic acid and phosphatidylethanolamine (18:2) in milk. Overall, untargeted metabolomics provided valuable information into metabolic pathways that may underlie FE in dairy ewes, with a special relevance of AA metabolism in determining this complex phenotype in the ovine. Further research is warranted to validate these findings.

Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation.

Small ruminants are susceptible to milk fat depression (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide individual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the ruminal responses of goats and sheep with varying degrees of MFD extent to improve our understanding of this complex syndrome. Our specific aims were to attempt to elucidate whether pre-existing variations in ruminal fermentation and biohydrogenation determine a higher tolerance or susceptibility to MFD, and whether the severity of MFD depends exclusively on the response to the diet. The trial was conducted with 25 does and 23 ewes fed a basal diet without lipid supplementation for 3 wk (control period). Then, 2% fish oil (FO) was added to the same diet for 5 additional weeks (MFD period). Based on the extent of the elicited MFD (i.e., the percentage variation between milk fat concentrations recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON-) animals were selected within each species. On the last day of each period, ruminal fluid samples were collected to examine fermentation parameters and fatty acid profiles. In general, the individual degree of MFD in sheep and goats did not seem to be predetermined by traits related to ruminal fermentation and biohydrogenation, including fatty acids that may serve as biomarkers of microorganisms. Regarding differences in the response to FO, the results suggest no link between MFD susceptibility and concentration of biohydrogenation intermediates such as trans-10-containing C18, C20, and C22 metabolites. The explanation for individual responses based on a shortage of ruminal acetate and 18:0 for mammary uptake also seems to be dismissed, based on the lack of variation in these compounds between RESPON+ and RESPON-. However, the concentration of unsaturated fatty acids provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3) was higher in the rumen of RESPON+ than RESPON- ewes and does. Thus, although further research is needed, the extent of biohydrogenation of these fatty acids might be associated with tolerance or susceptibility to MFD.

Insects as alternative feed for ruminants: comparison of protein evaluation methods.

BACKGROUND: The high dependence of intensive ruminant production on soybean meal and the environmental impact of this crop encourage the search for alternative protein-rich feeds. The use of insects seems promising, but the extent of their ruminal protein degradation is largely unknown. This parameter has major influence not only on N utilization efficiency but also on the environmental burden of ruminant farming. In addition, although assessing ruminal N degradation represents a key first step to examine the potential of new feeds, it is a challenging task due to the lack of a reference method. This study was conducted to investigate the potential of 4 insects (Tenebrio molitor, Zophobas morio, Alphitobius diaperinus and Acheta domesticus) as alternative protein sources for ruminants, using 3 methodologies: 1) a regression technique based on the in vitro relationship between gas production and ammonia-N concentration; 2) a conventional in vitro technique of batch cultures of ruminal microorganisms, based on filtering the incubation residue through sintered glass crucibles; and 3) the in situ nylon bag technique. The in vitro intestinal digestibility of the non-degraded protein in the rumen was also determined. Soybean meal was used as a reference feedstuff. RESULTS: Comparison of evaluation methods (regression, in vitro and in situ) did not allow to reliably select a single value of ruminal N degradation for the studied substrates, but all techniques seem to establish a similar ranking, with good correlations between methods, particularly between regression and in situ results. Regardless of the methodology, nitrogen from the 4 insects (with contents ranging from 81 to 112 g/kg of dry matter) did not show high ruminal degradation (41-76%), this value being always lower than that of soybean meal. Furthermore, the in vitro intestinal digestibility of non-degraded N was relatively high in all feeds (≥ 64%). CONCLUSION: Overall, these results support the potential of the 4 studied insects as alternative feedstuffs for ruminants. Among them, T. molitor showed the lowest and greatest values of ruminal N degradation and intestinal digestibility, respectively, which would place it as probably the best option to replace dietary soybean meal and increase the sustainability of ruminant feeding.

Elucidating genes and gene networks linked to individual susceptibility to milk fat depression in dairy goats.

Dietary supplementation with marine lipids modulates ruminant milk composition toward a healthier fatty acid profile for consumers, but it also causes milk fat depression (MFD). Because the dairy goat industry is mainly oriented toward cheese manufacturing, MFD can elicit economic losses. There is large individual variation in animal susceptibility with goats more (RESPO+) or less (RESPO-) responsive to diet-induced MFD. Thus, we used RNA-Seq to examine gene expression profiles in mammary cells to elucidate mechanisms underlying MFD in goats and individual variation in the extent of diet-induced MFD. Differentially expression analyses (DEA) and weighted gene co-expression network analysis (WGCNA) of RNA-Seq data were used to study milk somatic cell transcriptome changes in goats consuming a diet supplemented with marine lipids. There were 45 differentially expressed genes (DEGs) between control (no-MFD, before diet-induced MFD) and MFD, and 18 between RESPO+ and RESPO-. Biological processes and pathways such as "RNA transcription" and "Chromatin modifying enzymes" were downregulated in MFD compared with controls. Regarding susceptibility to diet-induced MFD, we identified the "Triglyceride Biosynthesis" pathway upregulated in RESPO- goats. The WGCNA approach identified 9 significant functional modules related to milk fat production and one module to the fat yield decrease in diet-induced MFD. The onset of MFD in dairy goats is influenced by the downregulation of SREBF1, other transcription factors and chromatin-modifying enzymes. A list of DEGs between RESPO+ and RESPO- goats (e.g., DBI and GPD1), and a co-related gene network linked to the decrease in milk fat (ABCD3, FABP3, and PLIN2) was uncovered. Results suggest that alterations in fatty acid transport may play an important role in determining individual variation. These candidate genes should be further investigated.

Individual differences in responsiveness to diet-induced milk fat depression in dairy sheep and goats.

Both sheep and goats can display very different individual degrees of milk fat depression (MFD), which might explain some apparent contradictions in the literature. Because the antilipogenic effect of certain fatty acids (FA) is the most likely origin of MFD, characterizing the milk FA profile of animals showing different degrees of MFD seems a helpful step to understand the physiological basis of the tolerance or susceptibility to the syndrome. Analyzing whether specific traits may predetermine a particular responsiveness would also be of relevance to meet this aim. However, information about these aspects is scant, not only in goats and sheep but in ruminants in general. This study was conducted with 25 Murciano-Granadina does and 23 Assaf ewes that were fed a total mixed ration without lipid supplementation for 3 wk (control period). Then, all animals received the same basal diet supplemented with 2% of fish oil (FO) for 5 additional weeks (MFD period). At the end of this second period, and on the basis of the extent of FO-induced decreases in milk fat concentration, the 5 most responsive (RESPON+) and the 5 least responsive (RESPON-) animals were selected within each species, 20 in total. Milk yield and composition, including a comprehensive FA profile, were examined at the end of each period. By design, between-group variation in milk fat concentration and yield was substantial, but no significant interaction with the effect of species was detected. Reductions in these 2 performance traits averaged 6% in RESPON- and 26% in RESPON+. Results do not allow suggesting that responsiveness to MFD would be clearly predetermined neither by the studied performance traits nor by milk FA profile, although a certain relationship with energy balance might exist. Furthermore, variations in ewes and does displaying different individual degrees of MFD may be associated with changes in certain candidate milk fat inhibitors, such as trans-10 18:1 and cis-9 16:1, whereas trans-10,cis-12 conjugated linoleic acid would only have a minor role in determining MFD severity. Alterations in the molar yield of de novo and preformed FA suggest relevant differences in the mechanisms underlying MFD in RESPON+ and RESPON-, with interspecies effects being observed only in more tolerant animals. Further research is still required to elucidate key determinants of responsiveness to MFD.

Effect of Dietary Supplementation with Lipids of Different Unsaturation Degree on Feed Efficiency and Milk Fatty Acid Profile in Dairy Sheep.

Lipids of different unsaturation degree were added to dairy ewe diet to test the hypothesis that unsaturated oils would modulate milk fatty acid (FA) profile without impairing or even improving feed efficiency. To this aim, we examined milk FA profile and efficiency metrics (feed conversion ratio (FCR), energy conversion ratio (ECR), residual feed intake (RFI), and residual energy intake (REI)) in 40 lactating ewes fed a diet with no lipid supplementation (Control) or supplemented with 3 fats rich in saturated, monounsaturated and polyunsaturated FA (i.e., purified palmitic acid (PA), olive oil (OO), and soybean oil (SBO)). Compared with PA, addition of OO decreased milk medium-chain saturated FA and improved the concentration of potentially health-promoting FA, such as cis-9 18:1, trans-11 18:1, cis-9 trans-11 CLA, and 4:0, with no impact on feed efficiency metrics. Nevertheless, FA analysis and decreases in FCR and ECR suggested that SBO supplementation would be a better nutritional strategy to further improve milk FA profile and feed efficiency in dairy ewes. The paradox of differences observed depending on the metric used to estimate feed efficiency (i.e., the lack of variation in RFI and REI vs. changes in FCR and ECR) does not allow solid conclusions to be drawn in this regard.

Determination of Cyclopropenoid Fatty Acids in Ewe Milk Fat by GC-MS after Intravenous Administration of Sterculic Acid.

Cyclopropenoid fatty acids (CPEFA), found in oilseeds from Malvaceae and Sterculiaceae, have been shown to interfere with the endogenous synthesis of several bioactive lipids of dairy fat, such as cis-9, trans-11 18:2 and cis-9 18:1, by inhibiting Δ9-desaturase. No previous study has reported the presence of sterculic acid in animal fat and its incorporation in tissues after its administration, due to the lack of a proper methodology. In the present research, a GC-MS method based on cold base derivatization to fatty acids methylesters was developed to determine CPEFA in ewe milk triglycerides, after infusing sterculic acid (0.5 g/day) to six lactating ewes. An alternative derivatization based on silanyzation followed by GC-MS analysis was also tested, showing its possible applicability when CPEFA are present in the form of free fatty acids. Sterculic acid was detected in ewe milk triglycerides, demonstrating its incorporation from the bloodstream into milk by the mammary gland. The mean transfer rate represented 8.0 ± 1.0% of the daily dose. This study provides, for the first time, the presence of sterculic acid in milk fat, supporting the importance of understanding its occurrence in vivo and encouraging further research to determine whether it can be present in foods, such as dairy products, obtained under practical farming conditions.

Conjugated linoleic acid (CLA)-induced milk fat depression: application of RNA-Seq technology to elucidate mammary gene regulation in dairy ewes.

Milk fat depression (MFD) is characterized by a reduction in the content of milk fat, presumably caused by the anti-lipogenic effects of rumen biohydrogenation intermediates, such as trans-10 cis-12 conjugated linoleic acid (CLA). In this study, RNA-Seq technology was used to help elucidate the mammary responses involved in CLA-induced MFD in lactating ewes. To this end, we compared the milk somatic cell transcriptome of ewes suffering from CLA-induced MFD with control ewes (i.e., those without MFD), as well as with ewes fed a diet supplemented with fish oil (FO-MFD) that we previously reported affects the mammary transcriptome. In the differential expression analysis between CLA-MFD and controls, we identified 1,524 differentially expressed genes (DEGs), whereas 653 were detected between CLA- and FO-MFD groups. Although this article focuses on lipid metabolism, CLA affected the expression of many genes related to other biological processes, especially immunity. Among the 55 genes shared by both MFD conditions, some genes linked to fatty acid synthesis, such as ACACA, AACS, ACSS2, or ACSS3, were downregulated. In addition, this study provides a list of candidate genes that are not usually considered in the nutrigenomics of MFD but that may act as key regulators of this syndrome in dairy ewes.

Conditions Associated with Marine Lipid-Induced Milk Fat Depression in Sheep Cause Shifts in the In Vitro Ruminal Metabolism of 1-13C Oleic Acid.

Shifts in ruminal oleic acid (OA) metabolism have received little research attention but recent studies have suggested their association with marine lipid-induced milk fat depression (MFD) in ewes and cows. Measurement of specific products of OA within the complex mixture of digesta lipids is however challenging. Therefore, this in vitro trial combined the isotopic labelling technique with the use of rumen inoculum from cannulated sheep fed a diet supplemented or not with 2% of fish oil (which has been demonstrated to cause MFD in dairy ruminants) to characterize the metabolism of OA in response to ruminal alterations associated with MFD. The products of 13C-OA after 24 h of incubation were analysed by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS). Numerous 13C-labeled 18:1 intermediates and oxygenated FA were detected and no elongation or desaturation of 13OA occurred. Diet supplementation with fish oil (i.e., MFD conditions) resulted in no unique metabolites of 13OA but in relevant changes in the relative contribution of specific metabolic pathways. The inhibition of 18:0 saturation caused by this treatment appeared largely attributable to increased oxygenated FA proportion, in particular the candidate milk fat inhibitor 10-oxo-18:0, and warrants further research on the association between MFD and oxygenated FA. Changes in the concentration of 13C-labeled trans 18:1 intermediates but not in cis 18:1, were also observed.

Elucidating fish oil-induced milk fat depression in dairy sheep: Milk somatic cell transcriptome analysis.

In this study, RNA sequencing was used to obtain a comprehensive profile of the transcriptomic changes occurring in the mammary gland of lactating sheep suffering from fish oil-induced milk fat depression (FO-MFD). The milk somatic cell transcriptome analysis of four control and four FO-MFD ewes generated an average of 42 million paired-end reads per sample. In both conditions, less than 220 genes constitute approximately 89% of the total counts. These genes, which are considered as core genes, were mainly involved in cytoplasmic ribosomal proteins and electron transport chain pathways. In total, 117 genes were upregulated, and 96 genes were downregulated in FO-MFD samples. Functional analysis of the latter indicated a downregulation of genes involved in the SREBP signaling pathway (e.g., ACACA, ACSL, and ACSS) and Gene Ontology terms related to lipid metabolism and lipid biosynthetic processes. Integrated interpretation of upregulated genes indicated enrichment in genes encoding plasma membrane proteins and proteins regulating protein kinase activity. Overall, our results indicate that FO-MFD is associated with the downregulation of key genes involved in the mammary lipogenesis process. In addition, the results also suggest that this syndrome may be related to upregulation of other genes implicated in signal transduction and codification of transcription factors.

Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid.

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11,cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11,cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore co-exist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11,cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11,cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11,cis-13 CLA and cis-9,trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11,cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue.