A post-GWAS confirming the genetic effects and functional polymorphisms of AGPAT3 gene on milk fatty acids in dairy cattle

Background: People are paying more attention to the healthy and balanced diet with the improvement of their living standards. Milk fatty acids(FAs) have been reported that they were related to some atherosclerosis and coronary heart diseases in human. In our previous genome-wide association study(GWAS) on milk FAs in dairy cattle, 83 genomewide significant single nucleotide polymorphisms(SNPs) were detected. Among them, two SNPs, ARS-BFGL-NGS-109493 and BTA-56389-no-rs associated with C18 index(P = 0.0459), were located in the upstream of 1-acylglycerol-3-phosphate O-acyltransferase 3(AGPAT3) gene. AGPAT3 is involved in glycerol-lipid, glycerol-phospholipid metabolism and phospholipase D signaling pathways. Hence, it was inferred as a candidate gene for milk FAs. The aim of this study was to further confirm the genetic effects of the AGPAT3 gene on milk FA traits in dairy cattle.Results: Through re-sequencing the complete coding region, and 3000 bp of 5′ and 3′ regulatory regions of the AGPA T3 gene, a total of 17 SNPs were identified, including four in 5′ regulatory region, one in 5′ untranslated region(UTR),three in introns, one in 3′ UTR, and eight in 3′ regulatory region. By the linkage disequilibrium(LD) analysis with Haploview4.1 software, two haplotype blocks were observed that were formed by four and 12 identified SNPs,respectively. Using SAS9.2, we performed single locus-based and haplotype-based association analysis on 24 milk FAs in 1065 Chinese Holstein cows, and discovered that all the SNPs and the haplotype blocks were significantly associated with C6:0, C8:0 and C10:0(P < 0.0001–0.0384). Further, with Genomatix, we predicted that four SNPs in 5′ regulatory region(g.146702957 G > A, g.146704373 A > G, g.146704618 A > G and g.146704699 G > A) changed the transcription factor binding sites(TFBSs) for transcription factors SMARCA3, REX1, VMYB, BRACH, NKX26, ZBED4, SP1, USF1, ARNT and FOXA1. Out of them, two SNPs were validated to impact transcriptional activity by performing luciferase assay that the alleles A of both SNPs, g.146704373 A > G and g.146704618 A > G, increased the transcriptional activities of AGPAT3 promoter compared with alleles G(P = 0.0004).Conclusions: In conclusion, our findings first demonstrated the significant genetic associations of the AGPAT3 gene with milk FAs in dairy cattle, and two potential causal mutations were detected.

Milk fatty acids profiles and milk production from dairy cows fed different forage quality diets

Thirty lactating Holstein cows were used to investigate the effects of different forages quality on milk fatty acids(FA) profiles and production. The cows were assigned to 3 dietary treatments(n = 10 per treatment) in a randomized block design with 3 repeated measures. They were fed the experimental diets for 90 d with 3 days of collection of samples for analysis at about 27 d intervals(samples were collected on days 28,29,30, 58, 59, 60.88, 89 and 90). The treatments were(DM basis): 1) mixed forages diet(MF) consisting of 3.7% Chinese wild rye, 26.7% corn silage and 23.4% alfalfa hay; 2) corn stalk diet 1(CS1) where corn stalk was used to formulate a similar chemical nutrient level to MF; 3) corn stalk diet 2(CS2) which used corn stalk to formulate a similar forage level to MF for the diet. Dry matter intake and BW were similar between treatments, but daily milk yield, milk fat and protein yield decreased(P < 0.05)in CS1 and CS2 compared with MF, with CS2 being the lowest(P < 0.05). In total FA of milk, the compositions of C18:1 c9, C18:3 and unsaturated FA increased(P < 0.05) in CS1 and CS2 compared with MF,and C18:0 and trans-C18:1 were trended to increase(P < 0.10), but C4:0-C16:0 were decreased(P < 0.05).Compared with cows fed CS2, cows receiving CS1 increased the compositions of C4:0 to C12:0 and C18:2(P < 0.05). The results suggests feeding corn stalk could produce a greater proportion of unsaturated fatty acid(UFA) in milk fat without resulting in milk fat depression(MFD) in mid lactation cows, but simply increasing the ratio of concentrate in low forages diets is not an effective way to increase milk fat synthesis and milk production.

Genetic Variants in the ELOVL5 but not ELOVL2 Gene Associated with Polyunsaturated Fatty Acids in Han Chinese Breast Milk

The present study was designed to examine the contributions of the fatty acid elongase （ELOVL） gene polymorphisms to the levels of polyunsaturated fatty acids （PUFAs） in breast milk. Two hundred and nine healthy Han Chinese mothers were included in the study. Carriers of minor alleles of SNPs （rs2397142 and rs9357760） in ELOVL5 were associated with higher levels of linoleic acid （LA）, dihomo-γ-linolenic acid （DGLA）, arachidonic acid （AA）, docosatetraenoic acid （DTA）, docosahexenoic acid （DHA）, while in rs209512 of ELOVL5 the carriers of minor alleles had lower levels of DTA compared to major homozygote alleles （P ranged from 0.004-0.046）, and genetically explained variability ranged from 3.2% for eicosapentaenoic acid （EPA） to 6.0% for LA. Our findings demonstrated that common variation in ELOVL5 gene encoding rate-limiting enzymes in the metabolism of PUFAs contribute to the PUFAs in breast milk.

Challenges in enriching milk fat with polyunsaturated fatty acids

Milk fatty acid composition is determined by several factors including diet. The milk fatty acid profile of dairy cows is low in polyunsaturated fatty acids, especially those of the n-3 series. Efforts to change and influence fatty acid profile with longer chain polyunsaturated fatty acids have proven challenging. Several barriers prevent easy transfer of dietary polyunsaturated fatty acids to milk fat including rumen biohydrogenation and fatty acid esterification. The potential for cellular uptake and differences in fatty acid incorporation into milk fat might also have an effect, though this has received less research effort. Given physiological impediments to enriching milk fat with polyunsaturated fatty acids, manipulating the genome of the cow might provide a greater increase than diet alone, but this too may be challenged by the physiology of the cow.

The effect of ensiled paulownia leaves in a high-forage diet on ruminal fermentation,methane production,fatty acid composition,and milk production performance of dairy cows

Background:The use of industrial by-products rich in bioactive compounds as animal feeds can reduce greenhouse gas production.Paulownia leaves silage(PLS)was supplemented to dairy cows'diet and evaluated in vitro(Exp.1;Rusitec)and in vivo(Exp.2,cannulated lactating dairy cows and Exp.3,non-cannulated lactating dairy cows).The study investigated the PLS effect on ruminal fermentation,microbial populations,methane production and concentration,dry matter intake(DMI),and fatty acid(FA)proportions in ruminal fluid and milk.Results:Several variables of the ruminal fluid were changed in response to the inclusion of PLS.In Exp.1,the p H increased linearly and quadratically,whereas ammonia and total volatile fatty acid(VFA)concentrations increased linearly and cubically.A linear,quadratic,and cubical decrease in methane concentration was observed with increasing dose of the PLS.Exp.2 revealed an increase in ruminal p H and ammonia concentrations,but no changes in total VFA concentration.Inclusion of PLS increased ruminal propionate(at 3 h and 6 h after feeding),isovalerate,and valerate concentrations.Addition of PLS also affected several populations of the analyzed microorganisms.The abundances of protozoa and bacteria were increased,whereas the abundance of archaea were decreased by PLS.Methane production decreased by 11%and 14%in PLS-fed cows compared to the control in Exp.2 and 3,respectively.Exp.3 revealed a reduction in the milk protein and lactose yield in the PLS-fed cows,but no effect on DMI and energy corrected milk yield.Also,the PLS diet affected the ruminal biohydrogenation process with an increased proportions of C18:3 cis-9 cis-12 cis-15,conjugated linoleic acid,C18:1 trans-11 FA,polyunsaturated fatty acids(PUFA),and reduced n6/n3 ratio and saturated fatty acids(SFA)proportion in milk.The relative transcript abundances of the 5 of 6 analyzed genes regulating FA metabolism increased.Conclusions:The dietary PLS replacing the alfalfa silage at 60 g/kg diet can reduce the methane emission and improve milk quality with greater proportions of PUFA,including conjugated linoleic acid,and C18:1 trans-11 along with reduction of SFA.

The effects of step-wise improvement of forage combination in total mixed rations on fatty acid profile in the rumen and milk of Holstein cows

Five lactating Holstein cows in a 5×5 Latin square experiment were fed five high-concentrate total mixed rations(TMRs) to investigate the effects of step-wise improvement of forage combination on ruminal and milk fatty acid profiles. The ratio of concentrate to forage was fixed as 61:39, and the step-wise improvement of forage combination was applied as: TMR1, a ration containing corn stover; TMR2, a ration containing corn stover and ensiled corn stover; TMR3, a ration containing ensiled corn stover and Chinese wild ryegrass hay(Leymus chinensis); TMR4, a ration containing the ryegrass hay and whole corn silage; TMR5, a ration containing the ryegrass hay, whole corn silage and alfalfa hay. The TMRs were offered to the cows twice daily at 0700 and 1900 h. The entire experiment was completed in five periods, and each period lasted for 18 days. Diurnal samples of rumen fluids were collected at 0100, 0700, 1300 and 1900 h(day 16); 0300, 0900, 1500 and 2100 h(day 17); and 0500, 1100, 1700 and 2300 h(day 18). The step-wise improvement of forage combination increased energy and crude protein contents and decreased fibre content. As a result, the step-wise improvement of forage combination increased dry matter intake and milk yield(P<0.05). The step-wise improvement increased dietary content of linolenic acid(C18:3 n-3), but did not alter dietary proportions of palmitic acid(C16:0), stearic acid(C18:0), oleic acid(C18:1 cis-9), linoleic acid(C18:2 n-6) and arachidic acid(C20:0). In response to the forage combination, ruminal concentration of C16:0, C18:2 n-6 and C18:3 n-3 linearly increased against their dietary intakes(P<0.10). The step-wise improvement increased milk contents of C10:0, C12:0, C14:0, C16:0, C18:2 n-6 and C18:3 n-3(P<0.10) and decreased milk contents of C18:0 and C18:1 cis-9(P<0.05). Milk yields of C16:0, C18:1 cis-9, C18:2 n-6 and C18:3 n-3 were linearly increased by the increase of these fatty acids in the rumen(R2≥0.79, P<0.05), and milk yields of C18:2 n-6 and C18:3 n-3 were also positively correlated with dietary intake of these fatty acids(R2≥0.85, P<0.05). The step-wise improvement increased the transfer efficiencies from feed to milk for C18:2 n-6 from 11.8 to 14.2% and for C18:3 n-3 from 19.1 to 22.3%. In a brief, along with the step-wise improvement of forage combination, more dietary linoleic and linolenic acids might escape microbial hydrogenation in the rumen and consequently accumulated in milk fat though these fatty acids were present in low concentrations in ruminal fluids. The step-wise improvement of forage combinations could be recommended as a dietary strategy to increase the transfer efficiency of linoleic and linolenic acids from feed to milk.

Increasing Conjugated Linoleic Acid Content in Milk and Cheese after Supplementing a Blend of Crude Soybean Oil Sediment Combined with Fish Oil to Grazing Dairy Cows

The aim of the work was to improve the healthy value of milk and cheese fatty acids (FA) by feeding a mix of crude soybean oil sediment (CSOS) combined with fish oil (FO) to grazing dairy cows. The CSOS is a by-product commonly discarded after oil extraction containing 3.3% moisture, 6% total ash and 70.7% oil, locally available, comparatively economic and easy to mix with other feed ingredients. The experiment lasted 55 days from September 30th to November 23th 2018 and was carried out at the dairy farm “Gacef” provider of milk to the dairy industrial plant “Capilla Del Señor” (CDS) located at the Villa María City, Córdoba Province, Argentine. A herd of 80 multiparous Holstein cows producing 24 kg-1 milk·cow-1·day-1 was used. The cows grazed an alfalfa and an oat pasture that represented about 47% of total dry matter (DM) intake supplemented at 8.5 kg DM·cow-1·day-1 with a total mixed ration (TMR) composed (DM basis) by cracked corn grain (35.18%), whole plant corn silage (31.98%), pelletized soyben meal (17.99%), the CSOS supplement (13.85%) and FO (0.99%). The TMR was supplied by halves after each milking time in groupal feeders yielding 1.4 kg·cow-1·day-1 of the CSOS and 0.1 kg·cow-1·day-1 of FO. Before the start of lipid supplementation, milk samples (5) were obtained from the farm-tank representing the standar or reference milk (Ref-Milk). After 21 days of supplementary lipid supply, additional milk samples (5) were obtained representing the modified milk (Mod-Milk). Milk samples were analyzed for chemical composition and milk FA profile. At each time, sufficient quantities of both (Ref- and Mod-Milk) were collected for manufacturing six types of cheeses. The results were analyzed through the Student-T test for independent observations. Oil supplementation did not modify (P > 0.05) the chemical composition of milk. Concentration of butyric acid (C4:0) in milk was not affected (P < 0.858). Concentration of total saturated FA (SFAs) in Ref-Milk averaged 58.83 g 100 g-1 FA and was decreased to 49.67 g 100 g-1 FA in Mod-Milk (P < 0.0001). Monounsaturated FA (MUFAs) increased (P < 0.001) from 32.03 g 100 g-1 FA in Ref-Milk to 38.13 g 100 g-1 FA in Mod-Milk (+19.07%) whereas polyunsaturated FA (PUFAs) increased (+36.1%) from 4.71 to 6.41 (P < 0.004). The Mod-Milk showed a significant (P < 0.002) reduction (-15.3% or 5.9 g 100 g-1 FA) for the total concentration of the potentially atherogenic fraction of milk FA (C12:0 to C16:0). The atherogenic index (AI) also decreased (P < 0.012) from 1.98 in Ref-Milk to 1.42 in Mod-Milk (-28.4%). Concentration of vaccenic acid (VA, trans-11 C18:1) in Mod-Milk averaged 7.77 g 100 g-1 FA which represented a 162 % increase (P < 0.0001) over that observed in Ref-Milk (2.95 g 100 g-1). Concentration of conjugated linoleic acid (CLA, cis-9, trans-11 C18:2) in Ref-Milk averaged 1.47 g 100 g-1 FA and showed an important increase (P < 0.002) in the Mod-Milk (3.86 g 100 g-1 FA, +163%). The omega 6/3 ratio resulted lower (P < 0.012) in the Ref-Milk (2.28) compared to the Mod-Milk (2.83). Milk and cheese FA composition were highly correlated (R2 = 0.99, P < 0.0001). The Mod-Cheeses showed similar results in AI, total concentration of SFAs, MUFAs and PUFAs compared to the milk of origin. Differences in FA composition between the cheeses made with the Ref- and Mod-Milk were equivalent to those described for milks. It is concluded that supplementation with a blend of CSOS supplement and FO was an effective way to improve the healthy value of dairy products by reducing contents of SFAs, atherogenic FAs and the atherogenicity index with a concomitant increase in VA and CLA. Modifications induced in the Mod-Milk were recovered in the Mod-Cheeses. The results obtained may help to reduce saturated fat intake and fight or prevent incidence of non-communicable, cardiovascular and chronic diseases.

Effects of total replacement of corn silage with sorghum silage on milk yield,composition, and quality

Background： In the last years, difficulties occurring in corn cultivation（i.e., groundwater shortages, mycotoxin contamination） have been forcing dairy farmers to consider alternative silages. Some experiments conducted on lactating cows have proven that the total replacement of corn silage with sorghum silage did not reduce milk yield.However, this kind of substitution involves supplementing sorghum-based diets with grains, to compensate for the lower starch content of sorghum silage compared to corn silage. Change of silage type and inclusion of starch sources in the diet would influence rumen fermentations, with possible effects on milk composition（i.e., fatty acid profile） and coagulation properties. A worsening of milk coagulation properties would have a negative economic impact in Italy, where most of the milk produced is processed into cheese.This study was designed to compare milk composition and quality, with emphasis on fatty acid profile and coagulation properties, in dairy cows fed two diets based on corn or sorghum silage.Results： The sorghum diet reduced milk yield（P = 0.043） but not 4% fat corrected milk（P = 0.85）. Feeding sorghum silage did not influence milk contents of protein（P = 0.07） and lactose（P = 0.65）, and increased fat content（P = 0.024）.No differences emerged for milk concentrations of saturated（P = 0.61） and monounsaturated fatty acids（P = 0.50）,whereas polyunsaturated fatty acids were lower（P 〈 0.001） for the sorghum diet. Concentrations of n-6（P 〈 0.001） and n-3 fatty acids（P = 0.017） were lower in milk of cows fed the sorghum diet. Milk coagulation properties did not differ between the two diets, except the ＂a30＂（the curd firmness, expressed in mm, 30 min after rennet addition）, that was lower（P = 0.042） for the sorghum diet.Conclusions： Feeding a forage sorghum silage, properly supplemented with corn meal, as total replacement of corn silage maintained milk composition and did not influence negatively milk coagulation properties, which have a great economic relevance for the Italian dairy industry. Thus, silages obtained from forage sorghums could have a potential as substitute of corn silages in dairy cow diets.

Sainfoin(Onobrychis viciifolia)silage in dairy cow rations reduces ruminal biohydrogenation and increases transfer efficiencies of unsaturated fatty acids from feed to milk

The effects of replacing grass silage by sainfoin silage in a total mixed ration(TMR)based diet on fatty acid(FA)reticular inflow and milk FA profile of dairy cows was investigated.The experiment followed a crossover design with 2 dietary treatments.The control diet consisted of grass silage,corn silage,concentrate and linseed.In the sainfoin diet,half of the grass silage was replaced by a sainfoin silage.Six rumen cannulated lactating multiparous dairy cows with a metabolic body weight of 132.5±3.6 kg BW^0.75,214±72 d in milk and an average milk production of 23.1±2.8 kg/d were used in the experiment.Cows were paired based on parity and milk production.Within pairs,cows were randomly assigned to either the control diet or the sainfoin diet for 2 experimental periods(29 d per period).In each period,the first 21 d,cows were housed individually in tie-stalls for adaptation,then next 4 d cows were housed individually in climate-controlled respiration chambers to measure CH4.During the last 4 d,cows were housed individually in tie stalls to measure milk FA profile and determine FA reticular inflow using the reticular sampling technique with Cr-ethylenediaminetetraacetic acid disodium salt dihydrate(EDTA)and Yb-acetate used as digesta flow markers.Although the dietary C18:3n-3 intake was lower(P=0.025)in the sainfoin diet group,the mono-unsaturated FA reticular inflow was greater(P=0.042)in cows fed the sainfoin diet.The reticular inflow of trans-9,trans-12-C18:2 and cis-12,trans-10 C18:2 was greater(P≤0.024)in the sainfoin diet group.The cows fed sainfoin diet had a lower(P≤0.038)apparent ruminal biohydrogenation of cis-9-C18:1 and C18:3n-3,compared to the cows fed the control diet.The sainfoin diet group had greater(P≤0.018)C18:3n-3 and cis-9,cis-12-C18:2 proportions in the milk FA profile compared to the control diet group.Transfer efficiencies from feed to milk of C18:2,C18:3n-3 and unsaturated FA were greater(P≤0.0179)for the sainfoin diet.Based on the results,it could be concluded that replacing grass silage by sainfoin silage in dairy cow rations reduces ruminal C18:3n-3 biohydrogenation and improves milk FA profile.