Fluorometric determination of isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP+ dependent) in ruminant milk.

The enzyme isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP+ dependent) located in the mammary cell cytosol mediates the synthesis of the majority of reducing equivalents for the energetically demanding milk fat and cholesterol synthesis in mammary cell cytosol. The present article presents a novel fluorometric method for quantification of the activity of this enzyme (IDH) in ruminant milk without pretreatment of the sample. Further, 493 goat milk samples - harvested before, during and after a nutritional restriction - were analysed for IDH activity i) with addition of extra substrate (isocitrate), and ii) with the intrinsic isocitrate solely. The IDH activity ranged from 0.22 to 15.4 units [nano moles product/(ml * min)] (un-supplemented) and from 0.22 to 45.6 units (isocitrate supplemented). The IDH activity increased considerably in milk during the nutritional restriction period concomitant with the increase in the metabolite isocitrate concentration and somatic cell count and returned to the initial level shortly after restriction period. The present 'high through-put' analytical method may be beneficial in future studies to phenotype modifications in mammary energy metabolism and milk fat synthesis, for which IDH activity may be a biomarker.

Exploration of robustness indicators using adaptive responses to short-term feed restriction in suckling primiparous beef cows.

Animal robustness is a complex trait of importance for livestock production systems and genetic selection. Phenotyping is essential for evaluation of the adaptation of different genotypes to changing environments. This study tested an experimental framework to induce marked deviations in the adaptive responses of suckling beef cows and to identify relevant indicators of responses to characterise individual differences in the robustness of cows. The production and metabolic responses of primiparous suckling Charolais cows to two periods of feed restriction (FR, 50% of their net energy requirements) of different durations were monitored. After calving, 13 cows (aged 39 ±â€¯2 months, BW of 680 ±â€¯42 kg at calving) had ad libitum access to a diet composed of hay and supplemented with concentrate to meet their energy and protein requirements. Starting at 54 ±â€¯6 days postcalving, the cows underwent two periods of FR: 4 days of FR (FR4), which was followed by 17 days of ad libitum intake to study the recovery from FR4, and 10 days of FR (FR10), which was followed by 18 days of ad libitum intake to study the recovery from FR10. The milk yield (MY), BW, body condition score and plasma non-esterified fatty acid (NEFA), ß-hydroxybutyrate, glucose and urea concentrations were measured before, during and after each FR. Among all measured variables, the MY and NEFA concentrations showed the most significant changes in response to FR. A functional data analysis approach was applied to the MY and NEFA data to model the adaptive responses and extract quantifiable indicators of deviation and recovery. Linear correlations (P < 0.03-0.07) between FR4 and FR10 were found for some indicators describing MY and NEFA levels before and after FR. The overall repeatability of MY and NEFA responses between both FR accounted for 46% based on quartile analysis performed on average responses. Moreover, the variance in both the MY and NEFA variables did not differ significantly between FR4 and FR10, despite a trend for higher variances in FR10. Altogether, (1) the calculated variables derived from the functional data analysis of the time patterns of the MY and NEFA accounted for the differences in the cow responses to FR, and (2) the animal responses appeared to show concordance between FR4 and FR10. In conclusion, short-term FR is a relevant framework for studying productive and metabolic adaptive responses in suckling cows and allows the identification of potential robustness indicators.

Milk metabolites and fatty acids as noninvasive biomarkers of metabolic status and energy balance in early-lactation cows.

The objective was to study the effects of week of lactation (WOL) and experimental nutrient restriction on concentrations of selected milk metabolites and fatty acids (FA), and assess their potential as biomarkers of energy status in early-lactation cows. To study WOL effects, 17 multiparous Holstein cows were phenotyped from calving until 7 WOL while allowed ad libitum intake of a lactation diet. Further, to study the effects of nutrient restriction, 8 of these cows received a diet containing 48% straw (high-straw) for 4 d starting at 24 ± 3 days in milk (mean ± SD), and 8 cows maintained on the lactation diet were sampled to serve as controls. Blood and milk samples were collected weekly for the WOL data set, and daily from d -1 to 3 of nutrient restriction (or control) for the nutritional challenge data set. Milk ß-hydroxybutyrate (BHB), isocitrate, glucose, glucose-6-phosphate (glucose-6P), galactose, glutamate, creatinine, uric acid, and N-acetyl-ß-d-glucosaminidase activity (NAGase) were analyzed in p.m. and a.m. samples, and milk FA were analyzed in pooled p.m. and a.m. samples. Average energy balance (EB) per day ranged from -27 MJ/d to neutral when cows received the lactation total mixed ration, and from -109 to -87 ± 7 MJ/d for high-straw (least squares means ± standard error of the mean). Plasma nonesterified FA concentration was 1.67 ± 0.13 mM and BHB was 2.96 ± 0.39 mM on the d 3 of high-straw (least squares means ± standard error of the mean). Milk concentrations of BHB, glucose, glucose-6P, glutamate, and uric acid differed significantly between p.m. and a.m. milkings. Milk isocitrate, glucose-6P, creatinine, and NAGase decreased, whereas milk glucose and galactose increased with WOL. Changes in milk BHB, isocitrate, glucose, glucose-6P, and creatinine were concordant during early lactation and in response to nutrient restriction. Milk galactose and NAGase were modulated by WOL only, whereas glutamate and uric acid concentrations responded to nutrient restriction only. The high-straw increased milk concentrations of FA potentially mobilized from adipose tissue (e.g., C18:0 and cis-9 C18:1 and sum of odd- and branched-chain FA (OBCFA) with carbon chain greater than 16; ∑ OBCFA >C16), and decreased concentrations of FA synthesized de novo by the mammary gland (e.g., sum of FA with 6 to 15 carbons; ∑ C6:0 to C15:0). Similar observations were made during early lactation. Plasma nonesterified FA concentrations had the best single linear regression with EB (R2 = 0.62). Milk isocitrate, Σ C6:0 to C15:0. and cis-9 C18:1 had the best single linear regressions with EB (R2 ≥ 0.44). Milk BHB, isocitrate, galactose, glutamate, and creatinine explained up to 64% of the EB variation observed in the current study using multiple linear regression. Milk concentrations of ∑ C6:0 to C15:0, C18:0, cis-9 C18:1, and ∑ OBCFA >C16 presented some of the best correlations and regressions with other indicators of metabolic status, lipomobilization, and EB, and their responses were concordant during early lactation and during experimental nutrient restriction. Metabolites and FA secreted in milk may serve as noninvasive indicators of metabolic status and EB of early-lactation cows.

Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

The objective was to investigate the effects of feed restriction on concentrations of selected milk metabolites in mid-lactation Holstein and Montbéliarde cows and to explore their correlations with energy balance and classic plasma and milk indicators of nutritional status. Eight Holstein and 10 Montbéliarde cows (165 ± 21 d in milk) underwent 6 d of feed restriction during which feed allowance was reduced to meet 50% of their net energy for lactation (NEL) requirements. The experiment was divided in 4 periods: control (CON; d -3 to -1), restriction (RES; d 1 to 6), wk 1 (W1; d 7 to 13), and wk 2 (W2; d 14 to 18) after refeeding at ad libitum intake. Intake, milk production, energy balance and plasma metabolites were used to validate the feed restriction model. Concentrations of 7 milk metabolites: ß-hydroxybutyrate (BHB), glucose, glucose-6-phosphate, isocitrate, glutamate, uric acid, and free amino groups were measured in morning milk samples, and fatty acids were measured in pooled p.m. and a.m. samples. Feed restriction induced a negative energy balance (-42.5 ± 4.4 MJ/d), increased plasma nonesterified fatty acids and BHB, and decreased plasma glucose concentrations. Feed restriction increased milk glucose-6-phosphate and isocitrate (+38% and +39%, respectively) and decreased milk BHB, glucose, glutamate, uric acid and free amino group concentrations (-20%, -57%, -65%, -42%, and -14%, respectively), compared with pre- restriction. Milk concentrations of medium-chain fatty acids (e.g., sum of C10 to C15) decreased and those of long chain (e.g., 18:0, cis-9 18:1) increased during restriction. Breed differences were not detected for the majority of variables. All studied milk metabolites were significantly correlated with energy balance (Spearman correlation = 0.48, 0.63, -0.31, -0.45, and 0.61 for BHB, glucose, glucose-6-phosphate, isocitrate, and glutamate, respectively). Milk glucose and glutamate showed the strongest correlations with plasma metabolites and milk FA associated with lipomobilization. These results suggest that milk metabolites may be used as noninvasive indicators of negative energy balance and metabolic status of dairy cows.

Deep RNA-Seq reveals miRNome differences in mammary tissue of lactating Holstein and Montbéliarde cows.

BACKGROUND: Genetic polymorphisms are known to influence milk production and composition. However, the genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNAs) regulate gene expression. Previous research suggests that the high developmental potential of the mammary gland may depend in part on a specific miRNA expression pattern. The objective of the present study was to compare the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, which have different mammogenic potentials that are related to differences in dairy performance. RESULTS: Milk, fat, protein, and lactose yields were lower in Montbéliarde cows than in Holstein cows. We detected 754 distinct miRNAs in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows using RNA-Seq technology, among which 738 were known and 16 were predicted miRNAs. The 25 most abundant miRNAs accounted for 90.6% of the total reads. The comparison of their abundances in the mammary glands of Holstein versus Montbéliarde cows identified 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, and 2 (miR-100 and miR-146b) were highly expressed. Among the most abundant miRNAs, miR-186 is known to inhibit cell proliferation and epithelial-to-mesenchymal transition. Data mining showed that 17 differentially expressed miRNAs with more than 20 reads were involved in the regulation of mammary gland plasticity. Several of them may potentially target mRNAs involved in signaling pathways (such as mTOR) and lipid metabolism, thereby indicating that they could influence milk composition. CONCLUSION: We found differences in the mammary gland miRNomes of two dairy cattle breeds. These differences suggest a potential role for miRNAs in mammary gland plasticity and milk component synthesis, both of which are related to milk production and composition. Further research is warranted on the genetic regulation of miRNAs and their role in milk synthesis.

Cow-level factors associated with subclinical hypocalcemia at calving in multiparous Jersey cows.

The objective of our study was to identify cow-level factors associated with subclinical hypocalcemia at calving (SCH) in multiparous Jersey cows. A total of 598 Jersey and 218 Jersey × Holstein crossbreed cows from 2 commercial dairy herds were enrolled in a retrospective cohort study. Blood samples to determine total Ca concentration were collected from the coccygeal vessels at 3 h 19 min (±2 h 33 min) after calving. We used 2 serum Ca concentration thresholds to define SCH: <2.00 mmol/L (SCH-2.00) and <2.12 mmol/L (SCH-2.12). We evaluated the association of cow-level factors with SCH with multivariable Poisson regression models. Variables evaluated for association with SCH were herd; parity (2, 3, and ≥4); breed; previous lactation length and 305-d mature-equivalent milk yield; previous lactation first test milk yield and last test somatic cell count; lengths of calving interval, gestation, dry, and close-up periods; body condition and locomotion scores at calving; calving ease; and calf sex for singletons. We categorized continuous variables into quartiles (≤25th percentile, interquartile range and ≥75th percentile). The prevalence of SCH among Jersey cows was 40 (SCH-2.00) and 64% (SCH-2.12). Jersey cows of higher parity had greater risk of SCH-2.00 and SCH-2.12. The risk of SCH-2.12 was higher after birthing male calves. We also found a tendency for previous lactation length and previous lactation 305-d mature-equivalent milk yield effect to affect risk of SCH-2.12. The risk of SCH-2.12 was lower for cows that had a previous lactation length shorter than the 25th percentile compared with cows that had a previous lactation length within the interquartile range. The risk of SCH-2.12 was higher for cows that had a previous lactation 305-d mature-equivalent milk yield below the 25th percentile compared with cows that had a previous lactation 305-d mature-equivalent milk yield above the 75th percentile. Also, Jersey × Holstein crossbreed was associated with increased risk of SCH-2.00. In the multivariable analysis, we observed no association between SCH and previous lactation first test milk yield; last test somatic cell count; lengths of calving interval, gestation, dry, and close-up periods; body condition and locomotion scores at calving; and calving ease. Our study identified parity, breed, calf sex, previous lactation length, and previous lactation 305-d mature-equivalent milk yield as cow-level factors associated with SCH in multiparous Jersey cows.

Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows.

The objective was to assess effects of experimentally induced undernutrition on responses to an intramammary lipopolysaccharide (LPS) challenge in early-lactation cows. Starting at 24 ± 3 d in milk, multiparous Holstein cows either received a ration containing 48% straw for 96 h to restrict nutrient intake (REST, n = 8) or were allowed ad libitum intake of a lactation diet (CONT, n = 9). After 72 h on diet or after an equivalent period for CONT, 50 µg of LPS (Escherichia coli 0111:B4) was injected into one healthy rear mammary quarter to induce an acute inflammation response. Blood samples were collected weekly until 7 wk of lactation, daily during feed restriction (or control), before and at 1, 2, 4, 6, 10, and 24 h relative to LPS injection. Foremilk quarter samples were collected before and at 4, 6, 10, and 24 h after LPS injection. Dry matter intake, milk yield, energy balance, plasma glucose, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate (BHB) concentrations did not differ between CONT and REST immediately before nutrient restriction in REST (least squares means at d -1 were 21.8, 39.0 kg/d, -2.5 MJ/d, and 3.78, 0.415, 0.66 mM, respectively) but were significantly altered at 72 h of nutrient restriction (9.8, 28.3 kg/d, -81.6 MJ/d, and 2.77, 1.672, and 2.98 mM, respectively), when the LPS challenge was performed. The rectal temperature increment from baseline values in response to LPS did not differ, but cortisol increment was greater and cortisol response area under the curve (AUC) tended to be greater [202 vs. 122 (ng/mL) × 10 h] for REST than CONT. No treatment differences were observed in foremilk IL-8, IL-1ß, tumor necrosis factor-α, and chemokine (C-X-C motif) ligand 3 concentrations in response to LPS injection. Composite milk somatic cell count per milliliter (6.919 × 106 vs. 1.956 × 106 cells/mL) and total number of somatic cells secreted in milk per day were greater for REST than CONT during the day following LPS. Plasma glucose, urea, and insulin concentrations increased after the LPS challenge, suggesting establishment of insulin resistance and modifications of glucose metabolism to support acute inflammation in both CONT and REST. Nonetheless, nutrient-restricted cows had delayed plasma insulin and glucose responses to LPS, smaller insulin AUC but greater glucose AUC compared with CONT, despite the limited nutrient availability to sustain an inflammation response. Undernutrition altered peripheral metabolic responses to an intramammary LPS challenge but had limited effects on selected indicators of inflammation response in early-lactation cows.

Effect of prophylactic oral calcium supplementation on postpartum mineral status and markers of energy balance of multiparous Jersey cows.

The effects of prophylactic oral Ca supplementation on blood mineral status and markers of energy balance were evaluated on 205 multiparous Jersey cows at a commercial dairy. Postpartum, cows were systematically assigned to control (n = 105) or oral Ca supplementation (CaOS; 50 to 60 g of Ca as boluses; n = 100) at 0 and 1 d in milk (DIM). Blood samples for analysis of serum minerals (Ca, P, Mg, K, Na, Fe, Zn, and Cu) were collected before and 1 h after treatment at 0 and 1 DIM, and at 2 DIM. Urine pH was measured immediately before and 1 h after treatment administration (n = 96). A subset of 74 cows was evaluated for plasma glucose and fatty acid concentrations at 0, 1, and 2 DIM. Cows were classified according to their initial calcemic status (Ca status) as normocalcemic (NC; serum Ca >2.12 mmol/L) or subclinically hypocalcemic (SCH; serum Ca ≤2.12 mmol/L). Average serum Ca concentration was higher in CaOS than control cows (2.12 vs. 2.06 mmol/L); this treatment effect was higher for SCH [CaOS (2.03 mmol/L); control (1.89 mmol/L)] than NC cows [CaOS (2.22 mmol/L); control (2.22 mmol/L)]. The incidence of subclinical hypocalcemia was lower for CaOS than control cows (53 vs. 65%); however, at 2 DIM the prevalence of subclinical hypocalcemia tended to be higher for CaOS cows, mostly because it was higher for CaOS-NC than control-NC cows (70 vs. 25%). Urine pH was lower for CaOS than control cows (6.10 vs. 7.04). Lower serum Mg concentration was detected for CaOS-SCH (1.06 mmol/L) than for control-SCH (1.10 mmol/L) cows. Cows in the CaOS group had higher serum K (4.68 vs. 4.53 mmol/L), lower plasma glucose (2.97 vs. 3.10 mmol/L), and at 2 DIM higher plasma fatty acid concentrations (0.43 vs. 0.35 mmol/L) than control cows. Our results showed that postpartum serum Ca concentration increases with oral Ca supplementation, but calcemic status influenced treatment response. Future studies should evaluate the long-term implications on production and reproduction of oral Ca supplementation in Jersey cows.

Effects of abomasal infusion of nicotinic acid on responses to glucose and ß-agonist challenges in underfed lactating cows.

The objectives were to assess the use of nicotinic acid (NA) to chronically (i.e., 74 h) manipulate plasma nonesterified fatty acid (NEFA) concentrations in partially feed-restricted lactating cows, determine whether the reduction of plasma NEFA altered responses to i.v. glucose tolerance test (ivGTT) and whether NA would attenuate an acute lipolytic stimuli of a ß-agonist challenge (ivBAC). Eight lactating dairy cows [244 ± 31 d in milk; 696 ± 63 kg of body weight (BW)] were blocked by breed and body condition score (3.2 ± 0.4) and randomly assigned to a sequence of 2 treatments in a crossover design. Treatments were 74-h continuous abomasal infusion of NA solution (3mg/h per kg of BW) as an antilipolytic agent to decrease plasma NEFA concentrations or the same volume of water (200 mL/h), concomitant with partial feed restriction. From 0 to 74 h of each experimental period, cows were feed-restricted to 33% of the ad libitum intake recorded during the prior 5 d. An ivGTT (0.25 g/kg of BW of glucose i.v.) and an ivBAC (4 nmol/kg of BW of isoproterenol hydrochloride, i.v.) were performed at 48 and 72 h, respectively. Intake was 24.1, 8.2, 8.0, and 8.0 kg of dry matter/d before restriction, on d 1, 2 and 3, respectively. Nicotinic acid decreased plasma NEFA and increased insulin and glucose concentrations during feed restriction. Nicotinic acid also led to greater glucose and insulin response areas under the curve during ivGTT [glucose: 6,562 vs. 5,056 (mg/dL) × 180 min; insulin: 6,042 vs. 2,502 (µIU/mL) × 180 min] and ivBAC [glucose: 535 vs. 240 (mg/dL) × 120 min; insulin: 1,283 vs. 222 (µIU/mL) × 120 min], and enhanced NEFA area under the curve during ivBAC [45,521 vs. 22,862 (µEq/L) × 120 min]. Milk, fat, and protein yields (29.1, 1.2, and 0.93 kg on d -2, respectively) decreased to 17.9, 0.81, and 0.56 kg for control, and 11.5, 0.54, and 0.39 kg for NA on d 3, respectively. Nicotinic acid may have decreased production by inhibiting the supply of NEFA for energy and milk fat synthesis. Milk urea nitrogen was increased by NA on d 2 (12.8 vs. 19.1mg/dL) and d 3 (11.6 vs. 17.8 mg/dL), probably due to a greater reliance on mobilized amino acids. Somatic cell count was increased by NA on d 3 (187 vs. 848 × 1,000 cells/mL). Patterns of glucose and insulin concentration observed during 74 h of NA infusion reflect a state of insulin resistance, which contrasts with shorter-term responses in nonlactating cows. Data suggest that long-term supraphysiological infusion of NA affected intermediary metabolism beyond antilipolysis and did not inhibit acute lipolytic stimuli of ivBAC.

Physiological adaptations and ovarian cyclicity of Holstein and Montbéliarde cows under two low-input production systems.

The objective was to study milk production, body reserve mobilization, metabolic and hormonal profiles, and ovarian cyclicity of Holstein-Friesian (HOLS) and Montbéliarde (MONT) cows under two low-input dairy production systems with seasonal spring calving: an extensive (EXT; 12 HOLS and 12 MONT) based on permanent diversified grasslands and zero concentrate, and a semi-extensive (SEMI; 12 HOLS and 10 MONT) based on established temporary grasslands and up to 4 kg/day of concentrate. Individual measurements were performed between -4 and 12 weeks of lactation. Cows in EXT secreted less milk (22.1 v. 24.4 kg/day), protein (660 v. 755 g/day) and energy (67.7 v. 74.4 MJ/day), had greater plasma ß-hydroxybutyrate (BHBA) (0.97 v. 0.69 mM), lower glucose (59.0 v. 62.0 mg/dl) and IGF-1 (62 v. 71 ng/ml), lower milk fat concentration in fatty acids originating from de novo synthesis (e.g. ∑ 10:0 to 15:0) and greater concentration of those derived in part from mobilization of fat reserves (e.g. 18:0 and ∑>C16), and showed greater frequency of abnormal ovarian cycles compared with SEMI. Across production systems, HOLS produced more milk (24.7 v. 21.8 kg/day), protein (738 v. 674 g/day) and fat (939 v. 819 g/day), secreted more energy (75.1 v. 67.0 MJ/day), lost more body condition score (BCS) (1.41 v. 1.03) and reached a lower BCS nadir (1.12 v. 1.43), had greater plasma BHBA (0.91 v. 0.75 mM), lower insulin (15.9 v. 17.2 µIU/ml) and tended to have lower glucose (59.6 v. 61.4 mg/dl), had lower milk fat concentration in ∑ 10:0 to 15:0, tended to have higher ∑>C16 and tended to show more abnormal estrous cycles compared with MONT. Ultrasound measurements did not differentiate fat mobilization and were confounded by breed differences of skin thickness. The greater nutrient allowance in SEMI improved indicators of physiological status and ovarian function during early lactation compared with EXT, but did not attenuate body reserve mobilization because cows prioritized milk secretion. HOLS secreted more nutrients than MONT but lost more BCS, which negatively affected nutritional balance and tended to affect ovarian cyclicity during early lactation. Breed by system interactions were not observed except for a few variables.

Rapeseed or linseed in dairy cow diets over 2 consecutive lactations: effects on adipose fatty acid profile and carry-over effects on milk fat composition in subsequent early lactation.

During early lactation, milk fatty acid (FA) composition is influenced by diet, animal genetics, and the high availability of preformed FA from body-fat mobilization. Long-term prepartum dietary oilseed supplementation could, therefore, modify milk FA composition postpartum in the subsequent lactation through changes in adipose tissue (AT) FA profile. To test this hypothesis, measurements were made in 19 Holstein cows fed grass-based diets containing no additional lipid (control, CTL; n=4) or supplemented with extruded linseeds (EL; n=4), cold-pressed fat-rich rapeseed meal (FRM; n=6), or whole unprocessed rapeseeds (WR; n=5) over 2 consecutive lactations (yr 1 and 2) and 2 dry periods. Oilseed supplements were withdrawn from the diets 23 d before the calving of yr 3, following the end of the previous experimental periods in yr 1 to 2. Thereafter, all cows received a total mixed ration composed of grass silage, grass hay, and concentrates (forage:concentrate ratio of 70:30 on a dry-matter basis). Cows previously fed EL and WR had a lower milk fat content (6.32% for CTL and FRM vs. 5.46% for EL and WR) and yield (1.90kg/d for CTL and FRM vs. 1.61kg/d for EL and WR) during the first week of lactation. Treatment effects on milk fat content and yield did not persist into lactation wk 3 and 7. Whatever the week, EL and WR increased concentration of FA in milk synthesized de novo (i.e., carbon number ≤15; 17.1g/100g of FA for CTL and FRM vs. 22.2g/100g of FA for EL and WR) and decreased concentration and secretion of preformed FA (i.e., carbon number ≥17; 56.1g/100g of FA for CTL and FRM vs. 49.9g/100g of FA for EL and WR). Alterations in milk FA composition may be explained by the lower availability of mobilized FA for uptake by the mammary gland, as indicated by the lower plasma nonesterified FA concentrations for EL and WR compared with CTL and FRM. Prepartum EL feeding increased AT and milk concentration of 18:3n-3 (0.96 vs. 0.79g/100g of milk FA for EL and the other groups, respectively) and intermediates of ruminal 18:3n-3 biohydrogenation. In contrast, FRM increased AT and milk concentration of ruminal cis-9 18:1 biohydrogenation intermediates. However, EL and FRM supplements resulted in a similar profile of 18-carbon FA isomers in AT (yr 2) and milk (yr 3, 4-10 wk after removing oilseeds from the diet). In conclusion, results confirm that long-term feeding of oilseed supplements alter AT FA composition and may influence milk FA composition during periods of extensive body-fat mobilization such as early lactation.

Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows.

The objective was to study the effects of body condition score (BCS) at calving on dairy performance, indicators of fat and protein mobilization, and metabolic and hormonal profiles during the periparturient period of Holstein-Friesian cows. Twenty-eight multiparous cows were classed according to their BCS (0 to 5 scale) before calving as low (BCS ≤ 2.5; n=9), medium (2.75 ≤ BCS ≤ 3.5; n=10), and high (BCS ≥ 3.75; n=9), corresponding to a mean of 2.33, 3.13, and 4.17 points of BCS, and preceding calving intervals of 362, 433, and 640 d, respectively. Cows received the same diets based on preserved grass to allow ad libitum feed intake throughout the study, and lactation diet contained 30% of concentrate (dry-matter basis). Measurements and sampling were performed between wk -4 and 7 relative to calving. No significant effects were observed of BCS group on dry matter intake (kg/d), milk yield, BCS loss, plasma glucose, and insulin concentrations. The high-BCS group had the lowest postpartum energy balance and the greatest plasma concentrations of leptin prepartum, nonesterified fatty acids and ß-hydroxybutyrate postpartum, insulin-like growth factor 1, and milk fat content. Milk fat yield was greater for the high- than the low-BCS group (1,681 vs. 1,417 g/d). Low-BCS cows had the greatest concentration of medium-chain fatty acids (e.g., sum of 10:0 to 15:0, and 16:0), and the lowest concentration and secretion of preformed fatty acids (e.g., cis-9 18:1) in milk fat. Milk protein secretion was lowest in the low-BCS group, averaging 924, 1,051, and 1,009 g/d for low-, medium-, and high-BCS groups, respectively. Plasma 3-methylhistidine was greater in wk 1 and 2 postpartum compared with other time points, indicating mobilization of muscle protein. Plasma creatinine tended to be lower and the 3-methylhistidine: creatinine ratio was greater in low- compared with medium- and high-BCS cows, suggesting less muscle mass but more intense mobilization of muscle protein in lean cows. High-BCS cows were metabolically challenged during early lactation due to intense mobilization of body fat. Conversely, limited availability of body fat in low-BCS cows was associated with increased plasma indicators of body protein mobilization during the first weeks of lactation, and lower milk protein secretion. These results should be confirmed using an experimental approach where calving BCS variation would be controlled by design.

Rapeseed or linseed supplements in grass-based diets: effects on dairy performance of Holstein cows over 2 consecutive lactations.

The aim of this study was to evaluate the effects of long-term supplementation with different oilseeds rich in 18:1 cis-9 or 18:3n-3 fatty acids on dairy cow performance over 2 consecutive lactations. This trial involved 58 Holstein cows during the first year and 35 during the second year. During the first 5 wk of the first year, all of the cows were fed the same diet; after a 4-wk transition period, the cows received 1 of 5 treatments for 2 consecutive lactations, including the dry period. Their basal diet was supplemented or not with extruded linseeds or with different forms of rapeseeds: extruded seeds, cold-pressed fat-rich meal, or whole unprocessed seeds. Oilseed amount was calculated to provide 2.5 to 3.0% additional oil in ration dry matter. Cows were fed a grass-based diet (75% grass silage and 25% hay) during indoor periods and grazed during outdoor periods. For the first year of experimentation, oilseed supplementation had no effect on milk, fat, protein, and lactose yields, body weight, or body condition score compared with the control treatment (no oilseed supplementation). During the indoor period, extruded linseed tended to decrease dry matter intake (-1.5 kg/d), whereas all of the oilseed treatments decreased milk protein content without changing protein yield. Cold-pressed fat-rich rapeseed meal decreased milk protein content independently of the period (-0.29 and -0.19 g/100 g for indoor and outdoor periods, respectively), and whole unprocessed rapeseed increased milk fat content during the outdoor period (+0.53 g/100 g compared with the control treatment). During the second year of experimentation, the effects of oilseed supplementation during the outdoor period were similar to those observed during the first outdoor period, but the effects of oilseed supplementation differed between the 2 indoor periods. This was likely due to changes in forage quality and composition and percentage in the ration of the concentrate mixtures. Thus, the effects of oilseed supplementation depended on oilseed nature (rapeseed or linseed) and form (extruded seeds, cold-pressed fat-rich meal, or whole unprocessed seeds) in interaction with the type of basal diet (grass silage and hay or pasture) and the concentrate composition and percentage in the ration. Effects were stable during the first indoor period, repeatable between the 2 outdoor periods, and were similar to effects observed previously in short-term studies (1 to 3 mo).

Antilipolytic and lipolytic effects of administering free or ruminally protected nicotinic acid to feed-restricted Holstein cows.

The objectives were to determine effects of 12 hourly infusions of different quantities of nicotinic acid (NA) on plasma nonesterified fatty acid (NEFA; experiment 1) and whether longer (108 h) continuous infusions of NA could induce sustained reductions of plasma NEFA (experiment 2) in nonlactating, nongestating Holstein cows that were feed restricted. Experiment 1 was a 5×5 Latin square with 6-d periods and 9 recovery days between each period. Each period consisted of 5 d of partial feed restriction to increase plasma NEFA concentration. Treatments were abomasal infusions of 0, 0.25, 0.5, 1, or 3 mg of NA/h per kilogram of body weight (BW), infused as hourly boluses for 12 h, starting 4 d after initiation of partial feed restriction. Plasma NEFA was decreased for the highest dose: from 448 µEq/L to 138±75 µEq/L at 1 h after the first bolus of 3mg of NA/h per kilogram of BW. This initial reduction in plasma NEFA concentration was followed by an increase in concentration at 2, 3, and 4 h relative to initiation of infusions. Plasma NEFA then decreased to 243 µEq/L 6h after initiation of treatments and remained low until termination of infusions. A rebound in plasma NEFA concentration occurred at 3 and 4 h after termination of infusion for cows that received 3 mg of NA/h per kilogram of BW. Experiment 2 was a 5×5 Latin square with 7-d periods and 9 recovery days between each period. Each period consisted of 5 d of partial feed restriction to increase plasma NEFA concentration. Treatments were continuous abomasal infusion of 0, 0.5, 1, or 3 mg of free NA/h per kilogram of BW for 4.5 d starting at feed restriction or 0.5 mg of NA/h per kilogram of BW infused directly into the rumen in a form protected from microbial degradation. The ruminal administration of protected NA was initiated 2 d before abomasal infusions and initiation of feed restriction to establish steady postruminal delivery of NA by start of abomasal infusions. Plasma NEFA was approximately 70 µEq/L before initiation of feed restriction and increased to 509, 587, 442, 850, and 108 µEq/L at 4.5 d for cows that received 0, 0.5 (protected NA), 0.5 (free NA), 1, and 3 mg of NA/h per kilogram of BW, respectively. An antilipolytic response was achieved with the highest abomasal dose, which maintained plasma NEFA concentration lower than the control group. An increase in plasma NEFA concentration was observed after termination of infusions for cows that received 1 and 3 mg of NA/h per kilogram of BW. Plasma NEFA was 1,900 µEq/L at 4h after termination of infusion for cows receiving 1 mg of NA/h per kilogram of BW and 1,360 µEq/L at 5h after termination of infusion for cows receiving 3 mg of NA/h per kilogram of BW. In nongestating, nonlactating cows it is unlikely that a dose of NA exists that will reduce plasma NEFA concentration and prevent the rebound that occurs following termination of NA administration.

Effects of abomasal lipid infusion on liver triglyceride accumulation and adipose lipolysis during fatty liver induction in dairy cows.

The objective was to determine the effects of abomasal infusion of linseed oil on liver triglyceride (TG) accumulation and adipose tissue lipolysis during an experimental protocol for induction of fatty liver. Eight nonpregnant, nonlactating Holstein cows were randomly assigned to treatments in a replicated 4 x 4 Latin square design. Treatments were abomasal infusion of water (W), tallow (T), linseed oil (LO), or half linseed oil and half tallow (LOT) at a rate of 0.56 g/kg of body weight per day. Each experimental period consisted of a 4-d fast concurrent with administration of treatments into the abomasum in 6 equal doses per day (every 4 h). Cows were fed ad libitum for 24 d between periods of fasting and lipid infusion. Infusion of linseed oil (LO and LOT) increased alpha-linolenic acid (C18:3n-3) content in serum (12.2, 10.4, 4.2, and 4.6 g/100 g of fatty acids for LO, LOT, T, and W, respectively), but not in the nonesterified fatty acid (NEFA) fraction of plasma. Treatments had no effect on plasma NEFA concentrations. Abomasal infusion of lipid increased in vitro stimulated lipolysis in subcutaneous adipose tissue, compared with W (4,294, 3,809, 4,231, and 3,293 nmol of glycerol released x g(-1) tissue x 2 h(-1) for LO, LOT, T, and W, respectively), but there was no difference between fat sources. Hepatic TG accumulation over 4-d fast was 2.52, 2.60, 2.64, and 2.09 +/- 0.75 microg of TG/microg of DNA for W, LO, LOT, and T, respectively, which did not differ. Abomasal infusion of LO did not reduce liver TG accumulation, plasma NEFA concentration, or alter in vitro adipose tissue lipolysis when compared with T. These results contrast with a previous study involving i.v. infusion of lipid emulsion derived from LO. Discrepancies might be explained by the use of different administration routes and a relatively modest induction of liver TG accumulation in the current experiment.

Effects of abomasal infusion of linseed oil on responses to glucose and insulin in holstein cows.

The objective was to study the effects of abomasal infusion of linseed oil, a source rich in n-3 C18:3, on whole-body response to insulin (experiment 1) and on insulin antilipolytic effects during feed restriction (experiment 2). In experiment 1, eight nonlactating, non-gestating cows were assigned to a crossover design, fed to meet maintenance requirements, and infused abomasally with either linseed oil (LIN) or tallow (TAL) at a rate of 0.54 g/kg of body weight per d for 5.5 d. Infusions were performed every 8 h during the first 3 d of each period and every 4 h thereafter. Intravenous glucose tolerance tests (IVGTT) were performed on d 5 of each period, followed by i.v. insulin challenges (IC) 12 h later. In experiment 2, six nonlactating, nongestating cows were assigned to a replicated 3 x 3 Latin square design. The experimental protocol included a water (WTR) treatment and feeding was suspended on d 3, leading to 50 and 62 h of feed restriction before IVGTT and IC, respectively. Clearance of glucose during IVGTT and IC was not affected by treatments in either experiment. However, LIN had an insulin sensitizing effect in experiment 1, because a lower insulin concentration led to the same clearance of glucose as TAL. In experiment 1, plasma nonesterified fatty acid (NEFA) concentration was low, reflecting a postprandial state, but NEFA was greater for LIN than TAL during IVGTT (108 vs. 88 +/- 4 microEq/L) and IC (133 vs. 83 +/- 9 microEq/L). In experiment 2, insulin concentrations during IVGTT did not differ across treatments. Basal plasma NEFA concentration before IVGTT tended to be greater for LIN than for TAL (612 vs. 508 microEq/L). Plasma NEFA clearance rate during IVGTT was greater for LIN than for TAL (2.8 vs. 2.5%/min), leading to a shorter time to reach half NEFA concentration (25 vs. 29 min) and greater absolute value of NEFA response area under the curve [AUC; -64,150 vs. -46,402 (microEq/L) x 180 min]. Data suggest that LIN enhanced the antilipolytic effects of insulin. Yet, other factors could have been involved because plasma NEFA concentration before IVGTT was 104 muEq/L greater for LIN than TAL for unknown reasons.

Reduction of plasma NEFA concentration by nicotinic acid enhances the response to insulin in feed-restricted Holstein cows.

The objective was to investigate the relationship between elevated plasma nonesterified fatty acid (NEFA) concentration and insulin resistance in Holstein cows. Six nonlactating, nongestating, ruminally cannulated Holstein cows were blocked by body condition score and randomly assigned to a sequence of 2 treatments in a crossover design. Cows were offered legume and grass hay ad libitum supplemented with minerals and vitamins and were allowed free access to water and a trace mineralized salt block. Mobilization of body reserves was stimulated by withdrawing forage for 48 h before initiation of treatments. Treatments consisted of 11 hourly abomasal infusions of water (control) or nicotinic acid (NA; 6 mg/h per kg of body weight) as an antilipolytic agent. Infusions of NA decreased plasma NEFA concentration from 545 microEq/L to approximately 100 microEq/L within 2 h after initiation of treatments, and differences were maintained throughout infusions. Intravenous glucose tolerance test was performed 8 h after initiation of treatments and was followed by 3 h of blood sampling. The reduction of plasma NEFA concentration led to significantly greater glucose clearance rate (1.9 vs. 1.2%/min) and to decreased glucose half-life (37 vs. 58 min), time to reach basal concentration (81 vs. 114 min) and glucose response area under the curve during 180 min of sampling [6,942 vs. 10,085 (microIU/mL) x 180 min]. Enhanced glucose clearance was achieved when plasma NEFA was reduced by NA, despite lower insulin concentration (70.0 vs. 97.9 +/- 13.4 microIU/mL) and a tendency for smaller insulin response area under the curve during 180 min of sampling [7,646 vs. 12,104 +/- 2,587 (microIU/mL) x 180 min], reflecting an increased response to endogenous insulin. Based on literature, we do not expect NA to have altered glucose metabolism directly; therefore, this experiment demonstrates a cause and effect relationship between elevated NEFA and insulin resistance in Holstein cows.

The use of nicotinic acid to induce sustained low plasma nonesterified fatty acids in feed-restricted Holstein cows.

The objectives were to determine the effects of nicotinic acid (NA) on blood metabolites (experiment 1) and whether successive doses of NA could induce sustained reductions of plasma nonesterified fatty acids (NEFA; experiment 2) in feed-restricted, nonlactating Holstein cows. Experiment 1 was a single 4 x 4 Latin square with 1-wk periods. Each period consisted of 2.5 d of feed restriction to increase plasma NEFA and 4.5 d of ad libitum feeding. Treatments were abomasal administration of 0, 6, 30, or 60 mg of NA/kg of body weight (BW), given as a single bolus 48 h after initiation of feed restriction. Plasma NEFA concentration decreased from 546 microEq/L to 208 +/- 141 microEq/L at 1 h after the infusion of 6 mg of NA/kg of BW, and to less than 100 +/- 148 microEq/L at 3 h after the abomasal infusion of the 2 highest doses of NA. A rebound occurred after the initial decrease of plasma NEFA concentration. The rebound lasted up to 9 h for the 30-mg dose of NA, and up to 6 h for the 6-mg dose. Experiment 2 was a randomized complete block design with 3 treatments and 6 cows. Starting at 48 h of feed restriction, cows received 9 hourly abomasal infusions of 0, 6, or 10 mg of NA/kg of BW. Plasma NEFA concentrations decreased from 553 microEq/L +/- 24 immediately before the initiation of treatments to <100 microEq/L during hourly infusions of 6 or 10 mg of NA/kg. Data suggest that the maximal antilipolytic response was achieved with the lowest dose of NA. A rebound of NEFA started 2 to 3 h after NA infusions were terminated. In both experiments, the NEFA rebound period coincided with increases in insulin and no change or increased glucose concentrations, suggesting a state of insulin resistance induced by elevated NEFA. This model for reducing plasma NEFA concentration by abomasal infusions of NA can be used to study the metabolic ramifications of elevated vs. reduced NEFA concentrations. The data demonstrate potential benefits and pitfalls of using NA to regulate plasma NEFA and prevent lipid-related metabolic disorders.

Induction of hyperlipidemia by intravenous infusion of tallow emulsion causes insulin resistance in Holstein cows.

The objective was to test whether the induction of elevated blood nonesterified fatty acids (NEFA) by i.v. infusion of a tallow emulsion altered glucose tolerance and responsiveness to insulin in Holstein cows. Six non-lactating, nongestating Holstein cows were assigned to a crossover design. One cow was excluded before initiation of the experiment because of complications from mastitis. Treatments consisted of 11-h i.v. infusions of saline (control) or a 20% (wt/vol) triacylglycerol (TG) emulsion derived from tallow (tallow) to elevate plasma NEFA. Each period consisted of two 11-h infusions (INF1 and INF2), separated by 1 d in which cows were not infused. Intravenous glucose tolerance tests (IVGTT) and insulin challenges (IC) were performed 8 h after initiation of INF1 and INF2, respectively. The infusion of treatments continued during the 3 h of sampling for IVGTT and IC. Cows were fed every 4 h at a rate to meet energy requirements for 5 d prior to each period, and every 2 h during the first 8 h of infusions. Infusion of tallow induced hyperlipidemia by increasing plasma NEFA (295 +/- 9 vs. 79 +/- 7 microEq/L), serum TG (41.0 +/- 6 vs. 11.4 +/- 4.4 mg/dL), and glycerol (0.81 +/- 0.09 vs. 0.23 +/- 0.1 mg/dL) concentrations during INF1. During INF2, tallow treatment increased plasma NEFA (347 vs. 139 +/- 18 microEq/L), serum TG (20.8 +/- 4.6 vs. 13.1 +/- 2.3 mg/dL), and glycerol (0.88 +/- 0.04 vs. 0.31 +/- 0.02 mg/dL) concentrations. Induction of hyperlipidemia impaired glucose clearance during IVGTT, despite the greater endogenous insulin response to the glucose infusion, leading to a lower insulin sensitivity index [0.29 vs. 1.88 +/- 0.31 x 10(-4) min(-1)/(microIU/mL)]. Accordingly, hyperlipidemia impaired glucose clearance during IC (1.58 vs. 2.72 %/min), reflecting lower responsiveness to insulin. These data show that induction of hyperlipidemia causes insulin resistance in Holstein cows by impairing both sensitivity and maximum responsiveness to insulin. The induction of insulin resistance by TG, NEFA, or both may increase the availability of glucogenic nutrients to the periparturient dairy cow. Yet excessive elevation of NEFA may potentially lead adipocytes to become more insulin resistant, further increasing plasma NEFA concentration and the risk of metabolic disorders.