Comparison of AlphaLISA and RIA assays for measurement of wool cortisol concentrations.

Radioimmunoassay (RIA) methods have always represented a technique of choice for the determination of steroids in biological samples. The Amplified Luminescent Proximity Homogenous Assay-Linked Immunosorbent Assay (AlphaLISA) is now emerging as the new-generation immunoassay technology that does not require washing/separation steps. The aim of this study was to adapt the Perkin-Elmer's AlphaLISA kit for wool cortisol and compare it with a RIA wool cortisol assay. Wool from lambs, 35 at birth (A0) and 54 at two months old (A2), was collected and each extract was evaluated for wool cortisol concentrations (HCC) both by RIA and AlphaLISA immunoassay. The two methods showed good precision, sensitivity and specificity for determining HCC. Both methods were able to detect significant differences between the high and the low HCC assessed in lambs at A0 and A2 (P < 0.01). The HCC assessed with RIA were significantly higher than those assessed with AlphaLISA (P < 0.01). Moreover, the correlation between HCC measured using the AlphaLISA and RIA methods was strong (r = 0.878). The regression analyses show a constant and not proportional error. This could be due to the diversity in the dosage steps and to the diversity of the molecules used in the two methods. Results support the validity of using AlphaLISA as an alternative method to RIA for the quantification of cortisol in sheep wool and considering the performances showed it has a great potential to be further applied as an excellent tool to evaluate HCC in samples derived from animal species.

Meat quality traits and the expression of tenderness-related genes in the loins of young goats at different ages.

Goat meat is considered healthy because of its low fat content, but it is often rather tough. Tenderness is the most important attribute of quality during meat consumption and there is scarce information about the expression of genes involved in the meat tenderization process in goats. The aim of this trial was to assess certain meat quality traits and the expression, at the messenger RNA (mRNA) and protein levels, of specific genes involved in the tenderization process of the longissimus lumborum (LL) in young male goats (Capra hircus) at different ages. Samples of LL were collected at slaughter from 32 Alpine goats that were divided into three categories: 9 suckling kids (Sk) at 5.4±0.15 weeks of age, 16 chevons (Ch) at 17.1±0.55 weeks of age and 7 post-puberal goats (Pu) at 34.3±2.5 weeks of age. Animal and carcass variables (live weight gain, live weight, carcass weight and fat deposits) and quality traits of meat (lipid content, ultimate pH, color parameters, cooking loss and shear force) were determined. The mRNA abundances of calpain-1 (Capn1), calpain-2 (Capn2), calpastatin (Cast), caspase 3 (Casp3), caspase 9 (Casp9), αB-crystallin (Cryab), heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) were detected by quantitative PCR. Capn1, Cast, Cryab and Hsp27 protein expression was investigated by ELISA. The Sk group had the leanest carcasses. The meat of the Pu group was the darkest (P<0.05) and the toughest (P<0.05). The redness of meat increased with the age of the goats. The Sk group showed lower mRNA abundances for the Capn2/Cast ratio, Casp3, Cryab, Hsp27, Hsp40 and Hsp70 than the Pu group (P<0.05). Intermediate values were found for the Ch group. Similar results were highlighted for the protein expression of Cryab and Hsp27. The experiment acknowledged a differentiation of the experimental groups based on performance, carcass and meat characteristics, and the genes considered. Moreover, Sk and Pu groups, characterized by a different tenderness of their meat, were clearly discriminated by a different expression of the Hsp.

Effects of grazing cow diet on volatile compounds as well as physicochemical and sensory characteristics of 12-month-ripened Montasio cheese.

The aim of this study was to evaluate the effects of pasture type and cow feeding supplementation level on a 12-mo-ripened Montasio protected designation of origin (PDO) cheese, which is one of the most important PDO cheeses produced in northeast Italy. Cheeses were characterized for volatile compounds, color, mechanical variables, and sensory descriptors. Pasture type significantly affected most of the instrumental variables considered and, as a consequence, sensory properties were affected as well. Cheeses from the pasture characterized by a nutrient-rich vegetation type were higher in protein and lower in fat content. Furthermore, such cheeses, evaluated by a sensory panel, were more intense in color with a more pungent and less cow-like odor, in agreement with what found through instrumental analyses. Supplementation level resulted in less pronounced effects, limited to volatile compounds and texture properties, which were not detected by sensory analysis. The characterization of the 12-mo ripened Montasio cheese reported here is an important step for the valorization of this PDO product.

Polymorphism of fat metabolism genes as candidate markers for meat quality and production traits in heavy pigs.

High meat quality is required for dry-cured ham production, which quality depends on meat fat quantity and composition. The aim was to study the polymorphisms of six genes involved in fat metabolism, namely, Stearoyl-CoA desaturase (SCD), Diacylglycerol acyltransferase 1 and 2 (DGAT1 &DGAT2), Microsomal triglyceride transfer protein (MTTP), Fatty acid synthase (FASN) and Heart fatty acid binding protein (H-FABP) in two traditional (Large White or Duroc × (Landrace × Large White)) and two industrial hybrids (Goland and Danbred), which are used for dry-cured ham production. Significant associations of SCD and MTTP were found with carcass weight. DGAT2 was associated with back fat thickness and L* fat colour (objective white colour score). Several genes (DGAT2, MTTP &FASN) were associated with weight loss during salting, first step in dry-cured ham production, affecting final yield. Finally, MTTP was associated with shear force. Our findings suggest that the SCD, DGAT2, MTTP and FASN polymorphisms are associated with quality of heavy pig meat products.

Volatile compounds and sensory properties of Montasio cheese made from the milk of Simmental cows grazing on alpine pastures.

The aim of this study was to analyze the volatile compounds, physicochemical characteristics, and sensory properties of Montasio, a semicooked pressed cheese, produced from the milk of the dual-purpose Italian Simmental cows grazing on alpine pastures. A total of 72 cows grazing on 2 pastures, which differed in botanical composition (nutrient-rich pasture vs. nutrient-poor pasture), received 2 different levels of supplementation (3.0 vs 1.5 kg/head per day). The experimental cheeses were produced from whole, raw milk and ripened for 60 d. Sixty-one volatile compounds, including alcohols (11), aldehydes (6), ketones (10), lactones (2), esters (6), hydrocarbons (3), carboxylic acids (6), phenolic compounds (4), monoterpenes (7), sesquiterpenes (1), sulfur compounds (4), and amines (1), were detected. The main families in terms of relative weight appeared to be carboxylic acids, esters, and alcohols. A panel of trained assessors described the experimental cheeses as having an intense color; small and evenly distributed eyes; an intense odor and flavor of milk-sour, milk, and cow; and a tender and creamy texture. The pasture type affected the volatile fraction, particularly ketones, phenolic compounds, and terpenes, which are overall higher in nutrient-poor pastures. A slight effect on the sensory analyses, in particular the effect of the cow attribute on odor and flavor, was perceived by the panelists. The cheeses produced on nutrient-rich pasture had higher b* (yellowness) index. These results were consistent with the color evaluation of the sensory panel. In addition, the pasture affected some textural attributes (adhesivity, creaminess, and granules) as perceived by the panelists. Concentrate supplementation, which is required to meet the feeding requirements of grazing cows, had no clear effect on either the volatile compounds or the sensory properties of the cheeses. Thus, at least within levels of integration adopted, it is expected not to alter the organoleptic characteristics of this product.

Study of progesterone and cortisol concentrations in the Italian Friesian claw.

The present research was conducted to study progesterone and cortisol concentrations in the claw of cattle and to verify whether the cattle claw could be considered an efficient matrix to provide retrospective information regarding progesterone and cortisol concentrations related to pregnancy and peripartum periods. These 2 steroids are involved in hoof growth. The study was performed on 32 calves and 24 pregnant milking cows of the Holstein breed, which were clinically healthy and lacking any claw disorders. Samples of at least 0.5cm in thickness were taken from the sole. Progesterone and cortisol concentrations were determined by RIA. The cortisol concentration in the horny shoe of calves from 0 to 30 d of age was significantly higher than the concentration at 31 to 60 and 61 to 120 d of age. Conversely, the progesterone concentration showed no statistically significant difference in relation to age. The horn progesterone concentrations recorded in the milking dairy cows at 7 mo of pregnancy showed high variability in the horizontal sections of the sole (the individual coefficient of variation ranged between 0.09 and 1.11). In 6 cows, genuine extreme values (genuine outliers) of the progesterone level were found. Moreover, significant differences existed among the progesterone concentrations of the sole's transverse sections. We detected a significant positive correlation between the weight of the horn samples after freeze-drying and their weight after hydration. The cortisol and progesterone levels in soaked horn samples were found to be significantly lower than in the same dry samples. These results show that cortisol and progesterone can be measured in the cattle claw horn. The claws of mature dairy cows could not be used as a matrix to provide a retrospective measure of cumulative hormone secretion, whereas the analysis of the calves' claw horns showed retrospective hormonal information similar to hair samples.

Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers.

The aim of this study was to evaluate the hypothalamic-pituitary-adrenal (HPA) axis activity of Holstein-Friesian and crossbreed F1 heifers by analysis of the cortisol concentrations in hair samples. Cortisol, the primary hormone of the HPA axis, is the biological endpoint for the investigation of the HPA response. The study was conducted on 290 prepubertal heifers; 142 heifers were pure Holstein-Friesian and 148 were crossbreed F1 heifers obtained from the 3-way rotational system with Swedish Red and Montbéliarde breeds. Extraction was performed on the hair using methanol, and cortisol concentrations were determined by a radioimmunoassay method. Cortisol concentrations measured in regrown hair of crossbreed F1 heifers were significantly lower than those in hair of Holstein-Friesian heifers. This result helps us to better understand the differences in HPA activity and allostatic load between Holstein-Friesian and crossbreed F1 heifers and allows us to better assess the adaptability of these animals to the environment and the importance of crossbreed traits for profitability in dairy farming.

Effect of linseed addition on the expression of some lipid metabolism genes in the adipose tissue of young Italian Simmental and Holstein bulls.

The objective of this trial was to determine the effect of breed and long-term dietary linseed addition on composition of fatty acids and expression of some genes involved in the lipid metabolism within subcutaneous (s.c.) adipose tissue of young bulls. Italian Simmental and Holstein bulls (n=16/breed) were fed a corn silage-grass hay diet with or without 8% (DM basis) whole ground linseed. Inclusion of linseed, rich in α-linoleic acid (C18:3n-3), increased (P<0.05) the proportions of linolelaidic (C18:2n-6trans), γ-linolenic (C18:3n-6), C18:3n-3, and rumenic (cis9,trans11 conjugated linoleic acid) acids, as well as total n-3 fatty acid, total PUFA, and PUFA:SFA, but decreased (P<0.05) weight percentages of myristic (C14:0), pentadecanoic (C15:0), palmitic (C16:0), palmitelaidic (C16:1n-9trans), and margaric (C17:0) acids, along with n-6:n-3, in the s.c. fat of young bulls. Even though PUFA were similar (P≥0.23) between bull breeds, s.c. fat from Holstein bulls had greater (P<0.05) proportions of tridecylic (C13:0), myristoleic (C14:1) and palmitoleic (C16:1n-9cis) acids and a lower (P<0.05) proportion of margaric (C17:0) acid than s.c. fat from Simmental bulls. Feeding linseed decreased (P<0.05) the expression of stearoyl CoA desaturase (SCD) and the lipoprotein lipase (LPL) gene without affecting (P≥0.19) fatty acid synthase (FASN), leptin (LEP), and PPARγ2 mRNA in the s.c. fat of bulls; however, there was no effect of bull breed (P≥0.11) or interactive effect of breed and linseed (P≥0.23) on gene expression. Expression of PPARγ2 was positively correlated with SCD (r=0.454; P=0.01), LEP (r=0.500; P<0.01), and LPL (r=0.531; P<0.01) mRNA, indicating that PPARγ2 increases the expression of genes involved in lipogenesis.

Effect of whole linseed addition on meat production and quality of Italian Simmental and Holstein young bulls.

The effect of long term dietary linseed addition on performance, carcass characteristics and meat quality of Italian Simmental (IS) and Italian Holstein (HI) young bulls was investigated. Thirty-two animals were assigned to 4 groups following a factorial design: 2 breeds - IS and IH - ×2 diets - containing whole ground linseed (5-8% of DM) and control. IS had greater in vivo performance and carcass characteristics than IH. IS muscle had lower C14:0, C16:0, SFA, higher C18:2n-6 cis, PUFAn-6, PUFA and PUFA/SFA proportion than IH in phospholipids (PL) fraction. Linseed inclusion did not affect animal's performance and carcass characteristics. In muscle PL, linseed increased C20:0, C22:0, C23:0, C20:5n-3 and decreased C20:4n-6, PUFAn-6/n-3, PUFAn-6 concentration. Linseed decreased C14:0, C16:0 proportion in neutral lipids (NL) and increased total PUFAn-3, C18:3n-3 proportions both in NL and PL fraction. However, these differences were relatively low from a quantitative point of view.

Association analyses of single nucleotide polymorphisms in the LEP and SCD1 genes on the fatty acid profile of muscle fat in Simmental bulls.

The aim was to investigate the effect of the genetic polymorphisms of leptin (LEP) and stearoyl-CoA desaturase (SCD1) genes on the fatty acid (FA) composition of the muscle of 103 Simmental bulls. Ten single nucleotide polymorphisms (SNP) were detected in exons 2 and 3 of the LEP gene, two of them encoding non-synonymous mutations. Allelic substitution effects of all the SNP on 28 single fatty acids, monounsaturated (MUFA) and polyunsaturated (PUFA) and desaturation indexes were estimated. Both the SCD1 SNP, as well as three SNP of the leptin gene, affected, to different extents, the desaturation of FA into MUFA. Because it was previously proposed that leptin's metabolic action involves down-regulation of SCD1, it is possible that, beyond the mere additive effect of SCD1 gene on FA desaturation, the non-synonymous mutations in the leptin gene also contribute to the variability of FA composition in muscle fat.

Fatty acid composition and sensory properties of Italian Simmental beef as affected by gene frequency of Montbéliarde origin.

The aim of this study was to investigate the effect of Montbéliarde (Mb) gene frequency on fatty acid composition and sensory properties of Italian Simmental (IS) steaks (longissimus thoracis m.). Twenty-seven bulls belonging to three strains with different percentages of Mb genes: traditional (ISt), without Mb ascendants (ISt=0% Mb genes), cross-strain (ISmt=37.5-50% Mb genes), Montbéliarde strain (ISm=87.5-100% Mb genes) and balanced for stearoyl Co-A desaturase genotype were considered. ISt has the highest C20:4 n-6 (P<0.01), C22:4 n-6 (P<0.05) and total PUFA n-3 level (P<0.01), while ISt and ISmt have higher C18:3 n-3 (P<0.05) and slightly lower MUFA (P=0.08) than ISm. Sensory tests indicated that the three experimental groups can be differentiated; moreover, ISmt meat is perceived as less hard (P<0.01), less chewable (P<0.01) and less fibrous (P<0.05) than ISt meat.