Short communication: Genetic variability in the predicted microRNA target sites of caprine casein genes

The main goal of the current work was to identify single nucleotide polymorphisms (SNP) that might create or disrupt microRNA (miRNA) target sites in the caprine casein genes. The 3′ untranslated regions of the goat α_S1-, α_S2-, β-, and κ-casein genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively) were resequenced in 25 individuals of the Murciano-Granadina, Cashmere, Canarian, Saanen, and Sahelian breeds. Five SNP were identified through this strategy: c.*175C > T at CSN1S1; c.*109T > C, c.*139G > C, and c.*160T > C at CSN1S2; and c.*216C > T at CSN2. Analysis with the Patrocles Finder tool predicted that all of these SNP are located within regions complementary to the seed of diverse miRNA sequences. These in silico results suggest that polymorphism at miRNA target sites might have some effect on casein expression. We explored this issue by genotyping the c.*175C > T SNP (CSN1S1) in 85 Murciano-Granadina goats with records for milk CSN1S1 concentrations. This substitution destroys a putative target site for miR-101, a miRNA known to be expressed in the bovine mammary gland. Although TT goats had higher levels (6.25 g/L) of CSN1S1 than their CT (6.05 g/L) and CC (6.04 g/L) counterparts, these differences were not significant. Experimental confirmation of the miRNA target sites predicted in the current work and performance of additional association analyses in other goat populations will be an essential step to find out if polymorphic miRNA target sites constitute an important source of variation in casein expression.     

The polymorphisms of stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes and their association with the fatty acid profile of muscle and subcutaneous fat in Fleckvieh bulls

The previously reported genetic polymorphisms of the stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes were investigated in Fleckvieh bulls using the PCR-RFLP and AS-PCR methods, respectively. The genomic DNA was obtained from a total of 370 bulls. The frequencies of alleles A and V of the single nucleotide polymorphism in exon 5 of the SCD1 gene (SNP 878C>T) were 0.555 and 0.445, respectively. In the 84-bp Ins/Del polymorphism in intron 5 of the SREBP-1 gene, the frequency of the L allele (insertion) was markedly higher (0.920) than that of the S allele (deletion; 0.080). Fatty acid profile was determined in a total of 367 samples of muscle fat (MSF) and 150 samples of subcutaneous fat (SCF). The AA genotype of SCD1 polymorphism showed a lower content of C18:0 (P < 0.01) and higher contents of C14:1 cis-9 (P < 0.001) and C18:1 cis-9 (P < 0.05) in MSF compared to the VV genotype. As a result, the bulls with genotypes AA or AV had lower SFA (P < 0.01), higher MUFA (P < 0.05) and higher MUFA/SFA (P < 0.01) than VV animals. The results obtained for SCF were similar. The SREBP-1 polymorphism was associated with a higher content of C14:1 cis-9 (P < 0.01) in the LS compared to LL genotype in SCF. The results of this study demonstrated the existence of the polymorphisms in the SCD1 and SREBP-1 genes in the population of Fleckvieh cattle and their associations with the concentrations of several MSF and SCF fatty acids.     

Short communication: Heritability of milk fatty acid composition and stearoyl-CoA desaturase indices in dairy cows

Activity of stearoyl-CoA desaturase (SCD) in the mammary gland is important for determining the relative proportions of saturated and monounsaturated fatty acids in milk and the concentration of the conjugated linoleic acid isomer rumenic acid (RA; cis-9,trans-11 18:2). Previous studies identified a large degree of between-cow variation in SCD activity, which was consistent across diets and suggests a genetic influence. The objectives of this study were to quantify genetic and phenotypic variations in fatty acid concentrations and SCD indices in milk fat and to estimate their heritabilities in a population of United Kingdom dairy cows. Milk samples were collected from 2,408 daughters of 597 Holstein-Friesian sires on 325 commercial farms for determination of fatty acid profiles. Indices of SCD activity were calculated by expressing each SCD product (cis-9 14:1, cis-9 16:1, cis-9 18:1, and RA) as a proportion of the precursor plus product [e.g., SCDI₁₄ = cis-9 14:1/(14:0 + cis-9 14:1)]. For individual fatty acids, phenotypic variance was considerably greater than additive genetic variance, resulting in small and nonsignificant heritability estimates (± standard error) for all except 6:0 (h² = 0.27 ± 0.10), 8:0 (h² = 0.27 ± 0.09), 12:0 (h² = 0.13 ± 0.07), cis-9 14:1 (h² = 0.28 ± 0.10), and cis-9 18:1 (h² = 0.12 ± 0.07). Heritability estimates of desaturase indices were significant for SCDI₁₄ (h² = 0.38 ± 0.11), SCDI₁₈ (h² = 0.19 ± 0.09), and SCDI_RA (h² = 0.21 ± 0.09), but not for SCDI₁₆ (h² = 0.05 ± 0.06). This study provides evidence that additive effects are responsible for a significant proportion of the phenotypic variation in SCD activity in dairy cows. It is concluded that because heritability of desaturase indices is moderate and significant in many cases, these indices could be investigated further for use in future breeding programs to increase concentrations of monounsaturated fatty acids and RA while decreasing concentrations of saturated fatty acids in milk fat.     

Milk fatty acid unsaturation: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1)

With regard to human health aspects of milk fat, increasing the amount of unsaturated fatty acids in milk is an important selection objective. The cow's diet has an influence on the degree of unsaturation, but literature suggests that genetics also plays a role. To estimate genetic variation in milk fatty acid unsaturation indices, milk fatty acid composition of 1,933 Dutch Holstein Friesian heifers was measured and unsaturation indices were calculated. An unsaturation index represents the concentration of the unsaturated product proportional to the sum of the unsaturated product and the saturated substrate. Intraherd heritabilities were moderate, ranging from 0.23 ± 0.07 for conjugated linoleic acid (CLA) index to 0.46 ± 0.09 for C16 index. We genotyped the cows for the SCD1 A293V and DGAT1 K232A polymorphisms, which are known to alter milk fatty acid composition. Both genes explain part of the genetic variation in unsaturation indices. The SCD1 V allele is associated with lower C10, C12, and C14 indices, and with higher C16, C18, and CLA indices in comparison to the SCD1 A allele, with no differences in total unsaturation index. In comparison to the DGAT1 K allele, the DGAT1 A allele is associated with lower C10, C12, C14, and C16 indices and with higher C18, CLA, and total indices. We conclude that selective breeding can contribute to higher unsaturation indices, and that selective breeding can capitalize on genotypic information of both the SCD1 A293V and the DGAT1 K232A polymorphism.     

Near infrared reflectance spectroscopy predicts the content of polyunsaturated fatty acids and biohydrogenation products in the subcutaneous fat of beef cows fed flaxseed

This study examined the ability of near infrared reflectance spectroscopy (NIRS) to estimate the concentration of polyunsaturated fatty acids and their biohydrogenation products in the subcutaneous fat of beef cows fed flaxseed. Subcutaneous fat samples at the 12th rib of 62 cows were stored at − 80 °C, thawed, scanned over a NIR spectral range from 400 to 2498 nm at 31 °C and 2 °C, and subsequently analysed for fatty acid composition. Best NIRS calibrations were with samples at 31 °C, showing high predictability for most of the n−3 (R²: 0.81-0.86; RMSECV: 0.11-1.56 mg g^− 1 fat) and linolenic acid biohydrogenation products such as conjugated linolenic acids, conjugated linoleic acids (CLA), non-CLA dienes and trans-monounsaturated fatty acids with R² (RMSECV_, mg g^− 1 fat) of 0.85-0.85 (0.16-0.37), 0.84-0.90 (0.21-2.58), 0.90 (5.49) and 0.84-0.90 (4.24-8.83), respectively. NIRS could discriminate 100% of subcutaneous fat samples from beef cows fed diets with and without flaxseed.     

Meat quality,proximate composition and muscle fatty acid profile of young llamas (Lama glama) supplemented with hay or concentrate during the dry season

Thirty llamas were used to study the effect of a 90 day feed supplementation on meat quality, chemical composition and muscle fatty acid profile. Treatments were: GR = llama on native pasture until slaughter; GR + SH = like GR, but with overnight free access to barley/alfalfa hay; and GR + SC = like GR, but with overnight free access to a wheat bran/sorghum grain concentrate. The supplementation had no effect on postmortem pH and temperature decline in the Longissimus lumborum muscle (LLM), cooking losses nor Warner–Bratzler shear force values (P > 0.05). Meat from GR + SC llama had higher fat content in LLM (P < 0.05) compared to GR and GR + SH llama. Intramuscular fat from GR + SH llama showed higher (P < 0.01) proportions of polyunsaturated fatty acids, higher (P < 0.05) polyunsaturated fatty acids/saturated fatty acids and desirable fatty acids ratio, lower (P < 0.05) omega-6/omega-3 (n − 6/n − 3) ratio, and higher (P < 0.01) conjugated linoleic acid.     

Effects of dietary replacement of fish oil by conjugated linoleic acid on some meat quality traits of Pacific white shrimp Litopenaeus vannamei

This experiment was conducted to evaluate the effects of fish oil replacement by conjugated linoleic acid (CLA) on body proximate analysis and thiobarbituric acid-reactive substances, fat content, shear force and fatty acid composition in musculature of Pacific white shrimp (Litopenaeus vannamei). Graded levels of CLA (0%, 0.5%, 1% and 2%) were added to the basic diet of shrimp at the expense of fish oil. Results showed that fat content (p = 0.036) and shear force (p = 0.001) in shrimp musculature were enhanced with increasing dietary CLA inclusion. Fish oil replacement by CLA significantly promoted the incorporation of cis-9, trans-11 CLA (p = 0.0001) and trans-10, cis-12 CLA (p < 0.0001) into shrimp musculature; moreover, the polyunsaturated fatty acid was elevated (p = 0.020) and monounsaturated fatty acid was reduced by CLA inclusion (p = 0.024). It was concluded that replacement of fish oil by CLA could improve some meat quality traits of shrimp and 1% CLA was an appropriate amount.     

Effects of corn silage particle length and forage:concentrate ratio on milk fatty acid composition in dairy cows fed supplemental flaxseed

This study aimed to evaluate the effects of length of chop of corn silage and forage:concentrate ratio (F:C) on performance and milk fatty acid profiles in dairy cows supplemented with flaxseed. Our hypothesis was that decreasing forage particle length and F:C ratio would increase unsaturated fatty acid flow to the small intestine and subsequent transfer of these unsaturated fatty acids into milk. Eight Holstein cows (648.1 ± 71.5 kg body weight; 109.6 ± 43.6 days in milk) were used in a replicated 4 × 4 Latin square design with 21-d periods and a 2 × 2 factorial arrangement of dietary treatments. Dietary factors were: 1) F:C ratios (dry matter basis) of 55:45 and 45:55; and 2) corn silage particle lengths of 9.52 and 19.05 mm. All experimental cows received 1 kg of flaxseed to substitute for 1 kg of a rolled barley grain-based concentrate daily. Diets were fed twice daily as a total mixed ration. Corn silage particle length and F:C ratio had no effect on dry matter intake, milk yield, and milk composition; however, feeding short cut corn silage depressed milk protein yield. Significant particle size × F:C ratio interactions were observed for milk fat proportions of C_16:0, C_18:1cis-9, and C_18:2cis-9, trans-11 (a conjugated linoleic acid isomer). At short corn silage particle size, decreasing F:C ratio depressed milk fat proportion of C_16:0. Conversely, feeding short corn silage at high F:C ratio increased the proportion of C_18:1cis-9 and C_18:2cis-9, trans-11 in milk fat. The milk fat proportion of C_18:2trans-10, cis-12, a conjugated linoleic acid isomer that is associated with milk fat depression, was not affected by dietary treatment. Our results show that corn silage particle length and F:C ratio influence milk fatty acid profiles in dairy cows fed supplemental flaxseed as a source of polyunsaturated fatty acids.     

Effect of weaning status on lipids of Galician Blond veal: Total fatty acids and 18:1 cis and trans isomers

The effect of weaning at different ages (NW = not weaned, W5 = 5.5 months and W2 = 2 months) on fatty acids (FA) of the Longissimus thoracis (LT) muscle was studied in 36 Galician Blond (GB) calves. Total FA (TFA) were determined by gas chromatography (GC) and 18:1 isomers by a combination of reversed-phase high performance liquid chromatography (HPLC) and GC. NW group showed higher (P < 0.001) values of n-3 polyunsaturated FA (PUFA), conjugated linoleic acid (CLA) and 18:1trans-11 compared to LT from W5 and W2 calves. W2 calves showed the highest levels of n-6 PUFA (P < 0.01), 18:1trans-10 and 18:1trans-6/7/8 (P < 0.001). Generally, W5 calves had intermediate values for TFA and 18:1 isomers. As the suckling period was longer, GB milk and veal FA profiles became more similar, it seems that muscle FA were partially transmitted by the milk FA intake due to the persistence of the reticular grove closing reflex.     

Enrichment of the conjugated linoleic acid content of bovine milk fat by dry fractionation

This study investigated the effect of fat fractionation on the conjugated linoleic acid (cis-9, trans-11-C_18 : 2) content of bovine milk fat. Anhydrous milk fat was fractionated into hard and soft fractions using controlled cooling and agitation. Fractionation of milk fat pre-melted at 60°C using a temperature programme of 33–10°C and a cooling rate of 0.58°C h⁻¹ yielded a soft fraction containing 63.2% more conjugated linoleic acid (2.22 g 100 g⁻¹ FAME), which was also enriched in polyunsaturated fatty acids and vaccenic acid (trans-11-C_18 : 1) compared with the parent fat. Agitation following fractionation was found to have a negative effect on the conjugated linoleic acid content of the soft fraction. Refractionation of the soft fraction did not increase the yield of conjugated linoleic acid. The conjugated linoleic acid and trans fatty acid content of 26 selected food products ranging in milk fat content from 0 to 100% is reported. Conjugated linoleic acid concentrations ranged from 0 to 16.2 mg g⁻¹ fat and were generally lower than the trans fatty acid content which ranged from 0 to 155.7 mg g⁻¹ fat. Spreads containing vegetable oils contained higher trans fatty acid and lower conjugated linoleic acid contents than milk fat-containing products. This study highlights that a milk fat fraction enriched in conjugated linoleic acid may be achieved by dry fractionation.     

Effects of fatty acid oxidation products (green odor) on rumen bacterial populations and lipid metabolism in vitro

This study investigated the effects of green odor fatty acid oxidation products (FAOP) from cut grass on lipid metabolism and microbial ecology using in vitro incubations of rumen microorganisms. These compounds have antimicrobial roles in plant defense, and we hypothesized that they may influence rumen lipid metabolism. Further, they may partially explain the higher levels of conjugated linoleic acid cis-9, trans-11 in milk from cows grazing pasture. The first of 2 batch culture experiments screened 6 FAOP (1 hydroperoxide, 3 aldehydes, 1 ketone, and 1 alcohol) for effects on lipid profile, and in particular C₁₈ polyunsaturated fatty acid biohydrogenation. Experiment 2 used the most potent FAOP to determine effects of varying concentrations and identify relationships with effects on microbial ecology. Batch cultures contained anaerobic buffer, rumen liquor, and FAOP to a final concentration of 100 μM for experiment 1. Triplicates for each compound and controls (water addition) were incubated at 39°C for 6 h. The hydroperoxide (1,2-dimethylethyl hydroperoxide, 1,2-DMEH) and the long chain aldehyde (trans-2 decenal) had the largest effects on lipid metabolism with significant increases in C_18:0 and C_18:1trans and reductions in C_12:0, C_14:0, C_16:0, C_18:1cis, C_18:2n-6, C_18:3n-3, C_20:0 and total branch and odd chain fatty acids compared with the control. This was associated with significantly higher biohydrogenation of C₁₈ polyunsaturated fatty acid. In experiment 2, 1,2-DMEH was incubated at 50, 100, and 200 μM for 2, 6, and 24 h. Increasing 1,2-DMEH concentration resulted in a significant linear increase in C_18:1trans-10, trans-11, conjugated linoleic acid, and C_18:0 and a linear decrease in C_18:2n-6 and C_18:3n-3, although the scale of this response declined with time. Microbial profiling techniques showed that 1,2-DMEH at concentrations of 100 and 200 μM changed the microbial community from as early as 2 h after addition, though microbial biomass remained similar. These preliminary studies have shown that FAOP can alter fatty acid biohydrogenation in the rumen. This change was associated with changes in the microbial population that were detected through DNA and branched- and odd-chain fatty acid profiling approaches.     

Growth and carcass fatty acid composition of beef steers fed soybean oil for increasing duration before slaughter

Duration of soybean oil (SBO) supplementation needed to enhance carcass conjugated linoleic acid (CLA) and trans-vaccenic (TVA) content was examined using 96 beef steers (293.6 ± 3.9 kg) fed a 78% corn-based diet supplemented with SBO for 0, 77, 137, or 189 days before slaughter. Duration of SBO supplementation had no effect (P ? 0.15) on animal performance or carcass traits, nor (P ? 0.15), total, total saturated, or total polyunsaturated fatty acids of Longissimus dorsi (LD). Concentrations of CLA in LD were not affected (P ? 0.18) by SBO supplementation. Concentrations of monounsaturated fatty acids (MUFA) decreased linearly (P = 0.03) in LD, whereas TVA increased (P = 0.04) in adipose tissue and tended (P = 0.07) to increase in LD with increasing duration of SBO supplementation. Supplementing SBO to a concentrate-based diet may enhance TVA without impacting CLA, while reducing the MUFA content of lean beef.     

Production system and slaughter age effects on intramuscular fatty acids from young Tudanca bulls

Thirty-three young bulls from Tudanca local breed were used to investigate the effect of two production systems (semi-extensive vs. intensive) and two slaughter ages (12 vs. 14 months) on meat fatty acid (FA) composition. Longissimus thoracis from semi-extensive animals had a lower percentage of intramuscular fat (p ≤ 0.001), saturated FAs (p ≤ 0.05), trans-18:1 (p ≤ 0.001), n-6/n-3 ratio (p ≤ 0.001) and a higher percentage of branched chain FAs (p ≤ 0.001), polyunsaturated FAs (p ≤ 0.001) and conjugated linoleic acid (CLA) (p ≤ 0.001). Few differences were observed in FA composition between slaughter ages. Hence, meat from semi-extensive animals slaughtered at 12 or 14 months displayed a healthier FA profile from a consumer's point of view.     

Association of polymorphisms at DGAT1, leptin, SCD1, CAPN1 and CAST genes with color,marbling and water holding capacity in meat from beef cattle populations in Sweden

The objective of this study was to investigate the associations of single nucleotide polymorphisms (SNPs) at five candidate genes with meat pH, color, marbling and water holding capacity (WHC) in young bulls from five beef breeds (n = 243) in Sweden. The UASMS2 polymorphism at the leptin gene and the SNPs at the Stearoyl-CoA desaturase 1 gene (SCD1.878) and μ-calpain gene (CAPN1:c.947) were associated with variation in meat color traits after 6 days of exposure to air. The K232A polymorphism at the diacylglycerol O-acyltransferase 1 (DGAT1) gene and the CAPN1:c.947 SNP were associated with level of beef marbling. There was no association between the SNP at the calpastatin gene (CAST:c.155) and meat quality traits, nor was there any association of the tested SNPs with WHC traits and pH value.     

A comparison between Holstein-Friesian and Jersey dairy cows and their F1 hybrid on milk fatty acid composition under grazing conditions

The objective of this study was to investigate the effect of 2 breeds, Holstein and Jersey, and their F₁ hybrid (Jersey × Holstein) on milk fatty acid (FA) concentrations under grazing conditions, especially conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids because of their importance to human health. Eighty-one cows (27 per breed grouping) were allocated a predominantly perennial ryegrass pasture. Samples were collected over 2 periods (June and July). Breed affected dry matter intake and milk production and composition. Holstein cows had the highest dry matter intake (18.4 ± 0.40 kg of DM/d) and milk production (21.1 ± 0.53 kg of DM/d). Holstein and Jersey × Holstein cows had similar 4% fat corrected milk, fat yield, and protein yield; with the exception of fat yield, these were all higher than for Jersey cows. Milk fat concentration was highest for Jersey cows and lowest for Holstein cows, with the hybrid cows intermediate. Total FA and linolenic acid intake (1.09 ± 0.023 and 0.58 ± 0.012 kg/d, respectively) were highest for Holstein cows. In terms of milk FA, Holstein cows had higher contents of C14:1, cis-9 C18:1 and linoleic acid. In turn, Jersey and Jersey × Holstein cows had higher content of C16:0. Milk concentrations of neither the cis-9,trans-11 isomer of CLA nor its precursor, vaccenic acid, were affected by breed. Nevertheless, large variation between individual animals within breed grouping was observed for CLA and estimated Δ⁹-desaturase activity. There was some evidence for a negative heterotic effect on milk concentration of CLA, with the F₁ hybrid cows having lower concentrations compared with the mid parent average. Plasma FA profile did not accurately reflect differences in milk FA composition. In conclusion, there was little evidence for either breed or beneficial heterotic effects on milk FA content with human health-promoting potential, though significant within-breed, interanimal variation was observed.     

Uptake and utilization of trans octadecenoic acids in lactating dairy cows

Trans fatty acids (FA) arise in ruminant-derived foods as a consequence of rumen biohydrogenation and are of interest because of their biological effects and potential role in chronic human diseases. Our objective was to compare 2 trans FA, elaidic acid (EA; trans-9 18:1) and vaccenic acid (VA; trans-11 18:1), with oleic acid (OA; cis-9 18:1) relative to plasma lipid transport and mammary utilization for milk fat synthesis. Three ruminally cannulated, Holstein dairy cows, 259 ± 6 DIM (mean ± SEM), were randomly assigned in a 3 × 3 Latin square design. Treatments were a 4-d abomasal infusion of 1) OA (45.5 g/d), 2) EA (41.7 g/d), and 3) VA (41.4 g/d). Milk samples were collected at each milking and blood samples were collected at the start and end of each treatment period. The proportions of total plasma FA associated with each plasma lipid fraction at baseline (pretreatment) were 62.6 ± 0.6% phospholipids, 26.1 ± 0.6% cholesterol esters, 9.8 ± 0.4% triglycerides, and 1.5 ± 0.1% nonesterified fatty acids; these values were unaffected by treatment. There were striking differences in the FA composition of the individual plasma lipid fractions and in the distribution of specific 18-carbon FA among the lipid fractions. Infusion of treatment isomers caused their specific increase in the various plasma lipid fractions but had no effect on milk production variables, including milk fat yield and content. Transfer efficiency of infused OA, EA, and VA to milk fat averaged 65.5 ± 3.0%, 59.7 ± 1.5%, and 54.3 ± 0.6%, respectively. For the VA infusion, 24.6 ± 1.1% of the transfer was accounted for by the increased yield of cis-9, trans-11 conjugated linoleic acid in milk fat, consistent with its endogenous synthesis from VA via the mammary enzyme Δ⁹-desaturase. Notably, linoleic acid (18:2n-6) and linolenic acid (18:3n-3) accounted for 47.7% of total plasma FA, but only 2.6% of FA in milk. Overall, results demonstrate clear differences in plasma transport and mammary uptake and utilization of 18-carbon FA, and these relate to the location, orientation, and number of double bonds.