Effect of dietary vitamin E on rumen biohydrogenation pathways and milk fat depression in dairy cows fed high-fat diets

Six lactating Holstein cows were assigned to a replicated Latin square design to test the effect of dietary vitamin E on milk fat depression and on the increased production of milk trans-10 C18:1 classically observed when feeding high doses of unsaturated fatty acids with low-fiber diets. Two diets (linseed diet and linseed diet + 12,000 IU of vitamin E/d) were compared during 2 periods of 21 d. The linseed diet presented a forage-to-concentrate ratio of 50:50 and contained extruded linseed (1.86 kg/d) and linseed oil (190 g/d). It was conceived to favor the “trans-11 to trans-10 shift” (low structural value and high level of unsaturated fatty acids). Milk yield and protein content were not affected by the diets. Milk of cows fed the linseed diet presented the typical symptoms of milk fat depression associated with a shift in biohydrogenation pathways: low fat content and high level of trans-10 C18:1. However, the high dose of dietary vitamin E provided significantly increased milk fat content (by 17.93%) and yield (by 15.56%) and decreased trans-10 C18:1 content (by 47.06%). In addition, it managed to significantly increase the daily yields of vaccenic (by 102.56%) and rumenic acids (by 56.67%). However, the sequence of administration of vitamin E influenced its effect, as vitamin E seemed to be more active in limiting the “trans-11 to trans-10 shift” when it was incorporated in the diet simultaneously with the fat. Once the shift had occurred, the subsequent addition of vitamin E was no longer able to completely counteract this process.     

A canonical discriminant analysis to study the association between milk fatty acids of ruminal origin and milk fat depression in dairy cows

Although milk fat depression (MFD) has been observed and described since the beginning of the last century, all the molecular and biochemical mechanisms involved are still not completely understood. Some fatty acids (FA) originating during rumen biohydrogenation have been proposed as causative elements of MFD. However, contradictory results were obtained when studying the effect of single FA on MFD. An alternative could be the simultaneous evaluation of the effect of many FA using a multivariate approach. The aim of this study was to evaluate the relationship between individual milk FA of ruminal origin and MFD using canonical discriminant analysis, a multivariate technique able to distinguish 2 or more groups on the basis of a pool of variables. In a commercial dairy herd, a diet containing 26% starch on a DM basis induced an unintentional MFD syndrome in 14 cows out of 40. Milk yielded by these 14 animals showed a fat content lower than 50% of the ordinary value, whereas milk production and protein content were normal. The remaining 26 cows secreted typical milk fat content and therefore were considered the control group, even though they ate the same diet. The stepwise discriminant analysis selected 14 milk FA of ruminal origin most able to distinguish the 2 groups. This restricted pool of FA was used, as variables, in a run of the canonical discriminant analysis that was able to significantly discriminate between the 2 groups. Out of the 14 FA, 5 conjugated linoleic acid isomers (C18:2 trans-10,trans-12, C18:2 trans-8,trans-10, C18:2 trans-11,cis-13, C18:2 cis-9,cis-11, C18:2 cis-10,cis-12) and C15:0 iso were more related to the control group, whereas C18:2 trans-10,cis-12, C16:1 trans-6–7, C16:1 trans-9, C18:1 trans-6–8, C18:1 trans-9, C18:1 trans-10, C18:1 cis-11, and C18:3n-3 were positively associated with the MFD group, allowing a complete discrimination. On the basis of these results, we can conclude that (1) the shift of ruminal biohydrogenation from C18:1 trans-11 to C18:1 trans-10 seemed to be strongly associated with MFD; (2) at the same time, other C18:1 trans isomers showed a similar association; (3) on the contrary, conjugated linoleic acid isomers other than C18:2 trans-10,cis-12 seemed to be associated with a normal fat secretion. Results confirmed that MFD is the consequence of a combined effect of the outflow of many ruminal FA, which collectively affect mammary fat synthesis. Because the animals of the 2 groups were fed the same diet, these results suggested that factors other than diet are involved in the MFD syndrome. Feeding behavior (i.e., ability to select dietary ingredients in a total mixed ration), rumen environment and the composition of ruminal bacteria are additional factors able to modify the products of rumen biohydrogenation. Results of the present work confirmed that the multivariate approach can be a useful tool to evaluate a metabolic pathway that involves several parameters, providing interesting suggestions about the role of some FA involved in MFD. However, results about the MFD syndrome obtained in the present research require a deep molecular investigation to be confirmed.     

Alterations in ruminal bacterial populations at induction and recovery from diet-induced milk fat depression in dairy cows

Ten ruminally cannulated Holstein cows were used in a crossover design that investigated changes in ruminal bacterial populations in response to induction and recovery from diet-induced milk fat depression (MFD). Further, the effect on the ruminal microbiota of the cows with diet-induced milk fat depression inoculated with rumen contents from non-milk fat-depressed donor cows was evaluated. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber, high-starch, and high-polyunsaturated fatty acid diet (26.1% neutral detergent fiber, 28.1% starch, 5.8% total fatty acids, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. Induction was followed by a recovery phase, where all cows were switched to a high-fiber, low-starch, and low-polyunsaturated fatty acid diet (31.8% neutral detergent fiber, 23% starch, 4.2% total fatty acids, and 1.2% C18:2) and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Ruminal samples were collected at the end of induction (d 10) and during recovery (d 13, 16, and 28), separated to solid and liquid fractions, extracted for DNA, PCR- amplified for the V1-V2 region of the 16S rRNA gene, and analyzed for bacterial diversity. Results indicated that bacterial communities were different between fractions. In each fraction, differences were significant between the induction (d 10) and recovery (d 13, 16, and 28) periods; however, differences were less apparent with time during the recovery period. The MFD (d 10) was typified by a reduction in the relative sequence abundance of Bacteroidetes and an increase in the relative sequence abundance of Firmicutes and Actinobacteria across both fractions. At the genus level, relative sequence abundance of unclassified Lachnospiraceae, Butyrivibrio, Bulleidia, and Coriobacteriaceae were higher on d 10 and were positively correlated with trans-10,cis-12 CLA and the trans-10 isomer, suggesting their potential role in altered biohydrogenation reactions. A switch to the recovery diet resulted in a sharp increase in the Bacteroidetes lineages and a decrease in Firmicutes members on d 13; however, this shift appears to stabilize by d 28, indicating the restoration process for ruminal bacteria from an altered state is gradual and complex. Inoculation of 10% of rumen contents from non-MFD donor cows to MFD cows revealed this procedure had transient effects on only a few bacterial populations, and such effects disappeared after d 16 following cessation of inoculation. It can be concluded that alterations in milk FA profiles at induction are preceded by microbial alterations in the rumen driven by dietary changes.     

Changes in fermentation and animal performance during recovery from classical diet-induced milk fat depression using corn with differing rates of starch degradability

Diet-induced milk fat depression (MFD) is a multifactorial disorder that can be triggered by a variety of conditions. Feeding high amounts of starch and unsaturated fatty acids has been shown to reduce milk fat yield and composition, as well as alter ruminal biohydrogenation patterns. However, little is known about how starch degradability in the rumen influences recovery from diet-induced MFD and if production of milk fat–inhibiting isomers will persist following an episode of MFD. The objective of this study was to evaluate production performance and ruminal fermentation in cows recovering from MFD when corn with a low or high starch degradability is fed. Six ruminally fistulated Holstein cows were used in a crossover design with 2 periods. During each period, MFD was induced for 10 d by feeding a diet with low fiber, high starch, and high unsaturated fatty acid. The polyunsaturated fatty acid concentration of the diet during the induction phase was modified primarily through inclusion of soybean oil. Following induction, cows were switched to either a high degradable starch recovery diet (HDS) or a low degradable starch recovery diet (LDS) for 18 d. The 7-h starch degradability was 66.5% for LDS and 87.8% for HDS. Milk was collected every 3 d for component and fatty acid analysis. On d 0, 4, 7, 10, 16, 22, and 28 of each period, ruminal pH and rumen fluid were collected every 2 h. Milk fat yield and composition was reduced during MFD induction and progressively increased by day in both HDS and LDS during recovery. Dry matter intake was similar among treatments and increased steadily over time during recovery. Preformed fatty acids were greater for HDS-fed animals, and de novo fatty acid in milk fat was greater for LDS-fed animals. Milk trans-10 C18:1 tended to be greater for HDS, and trans-10,cis-12 conjugated linoleic acid was significantly greater for HDS. cis-9,trans-11 conjugated linoleic acid was not affected by starch degradability during recovery. Total volatile fatty acids, butyrate, and valerate tended to differ or differed with recovery treatment, but ruminal pH and ammonia concentration were unaffected. The HDS diet responded similarly to the LDS diet during recovery with regard to milk fat percentage, but milk and fat yield tended to consistently be lower in HDS. When considering approaches to ameliorate diet-induced MFD, the degradability of the starch within rations should be evaluated. Although animal performance was similar, some trans fatty acid isomers were persistent in the milk through the recovery phase with HDS-fed animals, suggesting that milk fat synthesis might be potentially inhibited and biohydrogenation pathways modified in the rumen following an episode of MFD.     

Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration

Milk fat depression (MFD) caused by intermediates of ruminal biohydrogenation commonly occurs in dairy cattle. The time course of recovery from MFD is important to mechanistic investigation and management of the condition. Nine cows were used in a repeated design, allowing analysis of recovery from diet-induced MFD. A high-fiber, low-oil diet was fed during the control and recovery periods, and a low-fiber, high-oil (LFHO) diet was fed during the induction period. Milk yield was not affected by treatment. Milk fat percentage and yield decreased progressively during induction and were lower by d 3 and 5, respectively. Milk fat concentration and yield increased progressively when cows were fed the recovery diet and were not different from control on d 19 and 15, respectively. Yield of de novo synthesized fatty acids (FA) decreased progressively during the induction period and was lower than that of controls by d 5. A biphasic response was seen for milk fat trans isomers, where trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid (CLA) were elevated initially and trans-10 C18:1 and trans-10,cis-12 CLA increased progressively during the induction period. A similar biphasic response was seen during recovery from MFD, with trans-10 C18:1 and trans-10,cis-12 rapidly decreasing initially and trans-11 C18:1 and cis-9,trans-11 CLA increasing slightly above control levels during the second phase. Recovery from diet-induced MFD occurs gradually with a short lag when dietary fiber and oil concentrations are corrected. This time course provides a framework to identify factors causing MFD and set expectations during recovery from MFD.     

Analysis of alpha-linolenic acid biohydrogenation intermediates in milk fat with emphasis on conjugated linolenic acids

Ruminal biohydrogenation of α-linolenic acid is not fully understood compared with that of linoleic acid. Some hypothetical intermediates, that is, conjugated isomers of α-linolenic acid (cis-9,trans-11,cis-15 and cis-9,trans-13,cis-15 18:3) have never been reported to occur in ruminant fat. Therefore, milk fat was analyzed using a combination of techniques to characterize α-linolenic acid biohydrogenation intermediates. Tandem off-line argentation thin-layer chromatography and high-resolution gas-liquid chromatography using a 120-m highly polar, open tubular capillary column coated with 70% cyanoalkyl polysiloxane equivalent material was used for quantification. Structural characterization of fatty acids was achieved by gas-chromatography mass-spectrometry after synthesis of specific azo-derivatives. This study confirmed that minute amounts of α-linolenic acid biohydrogenation intermediates are present in milk fat. Routes involved in biohydrogenation of linoleic and α-linolenic acids in the rumen and subsequent endogenous metabolism of related biohydrogenation products are discussed.     

Effect of unsaturated fatty acids and triglycerides from soybeans on milk fat synthesis and biohydrogenation intermediates in dairy cattle

Increased rumen unsaturated fatty acid (FA) load is a risk factor for milk fat depression. This study evaluated if increasing the amount of unsaturated FA in the diet as triglycerides or free FA affected feed intake, yield of milk and milk components, and feed efficiency. Eighteen Holstein cows (132 ± 75 d in milk) were used in a replicated 3 × 3 Latin square design. Treatments were a control (CON) diet, or 1 of 2 unsaturated FA (UFA) treatments supplemented with either soybean oil (FA present as triglycerides; TAG treatment) or soybean FA distillate (FA present as free FA; FFA treatment). The soybean oil contained a higher concentration of cis-9 C18:1 (26.0 vs. 11.8 g/100 g of FA) and lower concentrations of C16:0 (9.6 vs. 15.0 g/100 g of FA) and cis-9,cis-12 C18:2 (50.5 vs. 59.1 g/100 g of FA) than the soybean FA distillate. The soybean oil and soybean FA distillate were included in the diet at 2% dry matter (DM) to replace soyhulls in the CON diet. Treatment periods were 21 d, with the final 4 d used for sample and data collection. The corn silage- and alfalfa silage-based diets contained 23% forage neutral detergent fiber and 17% crude protein. Total dietary FA were 2.6, 4.2, and 4.3% of diet DM for CON, FFA, and TAG treatments, respectively. Total FA intake was increased 57% for UFA treatments and was similar between FFA and TAG. The intakes of individual FA were similar, with the exception of a 24 g/d lower intake of C16:0 and a 64 g/d greater intake of cis-9 C18:1 for the TAG compared with the FFA treatment. Compared with CON, the UFA treatments decreased DM intake (1.0 kg/d) but increased milk yield (2.2 kg/d) and milk lactose concentration and yield. The UFA treatments reduced milk fat concentration, averaging 3.30, 3.18, and 3.11% for CON, FFA, and TAG treatments, respectively. Yield of milk fat, milk protein, and 3.5% fat-corrected milk remained unchanged when comparing CON with the UFA treatments. No differences existed in the yield of milk or milk components between the FFA and TAG treatments. The UFA treatments increased feed efficiency (energy-corrected milk/DM intake), averaging 1.42, 1.53, and 1.48 for CON, FFA, and TAG treatments, respectively. Although milk fat yield was not affected, the UFA treatments decreased the yield of de novo (<16-carbon) synthesized FA (40 g/d) and increased the yield of preformed (>16-carbon) FA (134 g/d). Yield of FA from both sources (16-carbon FA) was reduced by the UFA treatments but to a different extent for FFA versus TAG (72 vs. 100 g/d). An increase was detected in the concentration of trans-10 C18:1 and a trend for an increase in trans-10,cis-12 C18:2 and trans-9,cis-11 C18:2 for the UFA treatments compared with CON. Under the dietary conditions tested, UFA treatments supplemented at 2% diet DM as either soybean FA distillate or soybean oil increased milk yield but did not effectively cause a reduction in milk fat yield, with preformed FA replacing de novo synthesized FA in milk fat. Further research is required to determine if the response to changes in dietary free and esterified FA concentrations is different in diets that differ in their risk for milk fat depression.     

Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression

Diet-induced milk fat depression (MFD) is a multifactorial condition resulting from the interaction of numerous risk factors, including diet fermentability and unsaturated fatty acids concentration, feed additives, and individual cow effects. 2-Hydroxy-4-(methylthio)butanoate (HMTBa) is a methionine analog that has been observed to increase milk fat in some cases, and interactions with MFD risk factors may exist. The objective was to evaluate the effect of HMTBa supplementation on milk fat synthesis in cows with different levels of milk production and fed diets with increasing risk of biohydrogenation-induced MFD. Sixteen high-producing cows (44.1 ± 4.5 kg of milk/d; mean ± SD) and 14 low-producing (31.4 ± 4.3 kg of milk/d) were used in a randomized block design. Treatments were unsupplemented control and HMTBa fed at 0.1% of diet dry matter (25 g/d at 25 kg of dry matter intake). The experiment was 70 d and included a 14-d covariate period followed by 3 phases whereby diets were fed with increasing risk of MFD to determine the interaction of treatment and diet-induced MFD. During the low-risk phase, the base diet was balanced to 33.5% neutral detergent fiber (NDF) and had no exogenous oil (28 d); during the moderate-risk phase, the diet was balanced to 31% NDF and contained 0.75% soybean oil (14 d); and, during the high-risk phase, the diet was balanced to 28.5% NDF and contained 1.5% soybean oil (14 d). An interaction of treatment, production-level, and dietary phase was observed. Low producing cows neither experienced substantial biohydrogenation-induced MFD nor a response in milk fat to HMTBa supplementation. In high-producing cows, HMTBa maintained higher milk fat concentration during the moderate- (2.94 vs. 3.49%) and high-risk (2.38 vs. 3.11%) phases. High-producing cows receiving HMTBa also had greater milk fat yield (0.94 vs. 1.16 kg/d) and lower trans-10 C18:1 (6.11 vs. 1.50) during the high-risk phase. In conclusion, HMTBa increased milk fat in situations with a high risk of biohydrogenation-induced MFD by decreasing absorption of alternate biohydrogenation intermediates.     

Short communication: Effects of molasses products on productivity and milk fatty acid profile of cows fed diets high in dried distillers grains with solubles

Previous research has shown that replacing up to 5% [of dietary dry matter (DM)] corn with cane molasses can partially alleviate milk fat depression when cows are fed high-concentrate, low-fiber rations containing dried distillers grains with solubles. The primary objective of this study was to determine whether dietary molasses alters milk fatty acid (FA) profile or improves solids-corrected milk yield in the context of a more typical lactation diet. A secondary objective was to assess production responses to increasing rumen-degradable protein supply when molasses was fed. Twelve primiparous and 28 multiparous Holstein cows (196 ± 39 d in milk) were blocked by parity and assigned to 4 pens. Pens were randomly allocated to treatment sequence in a 4 × 4 Latin square design, balanced for carryover effects. Treatment periods were 21 d, with 17 d for diet adaptation and 4 d for sample and data collection. Treatments were a control diet, providing 20% dried distillers grains with solubles (DM basis), 35% neutral detergent fiber, 30% starch, and 5% ether extract; a diet with 4.4% cane molasses replacing a portion of the corn grain; a diet with 2.9% molasses supplement containing 32% crude protein on a DM basis; and a diet with 5.8% (DM basis) molasses supplement. Animal-level data were analyzed using mixed models, including the fixed effect of treatment and the random effects of period, pen, period × pen interaction, and cow within pen to recognize pen as the experimental unit. Diets did not alter DM intake, milk production, milk component concentration or yield, feed efficiency (DM intake/milk yield), body weight change, or milk somatic cell count. Milk stearic acid content was increased by the diet containing 5.8% molasses supplement compared with the control diet and the diet containing 2.9% molasses supplement, but the magnitude of the effect was small (12.27, 11.75, and 11.69 ± 0.29 g/100 g of FA). Production data revealed a dramatic effect of period on milk fat content and yield. Milk fat content decreased during the course of the experiment (least squares means = 3.16, 2.81, 2.93, and 2.64 ± 0.09% for periods 1 to 4, respectively), as did milk fat yield (1.20, 1.03, 0.98, and 0.79 ± 0.05 kg/d). Exchanging molasses-based products for corn at 2.9 to 5.8% of dietary DM did not influence productivity and had minute effects on milk FA profile. The limited responses in this study may have been influenced by dietary unsaturated FA content or the advancing stage of lactation of cows in the study.     

Short communication: Effect of antioxidant supplementation on milk production,milk fat synthesis,and milk fatty acids in dairy cows when fed a diet designed to cause milk fat depression

This study evaluated the effect of a blend of synthetic antioxidants on the yield of milk and milk components and milk fatty acid composition in dairy cows fed a diet designed to cause milk fat depression (MFD). We hypothesized that supplementing a synthetic antioxidant to diets with a high rumen unsaturated fatty acid load (RUFAL) would decrease the severity of MFD. Sixteen lactating Holstein cows (163 ± 47 d in milk), in a crossover design with two 21-d periods, were fed a corn silage and grass silage-based diet containing 15% distillers grains. The diet contained 34% neutral detergent fiber, 18% crude protein, 26% starch, and 4.3% total fatty acids (dry matter basis). Cows were fed the diet without supplementation (control; CON) or supplemented with 0.02% (dry matter basis) of a synthetic antioxidant (AOX; Agrado Plus, Novus International Inc., St. Charles, MO). Dry matter intake and milk yields were recorded daily. Milk samples were collected at the start of the study for baseline values and the end of each period (d 20–21) and analyzed for milk components and fatty acid composition. Dry matter intake and milk yield were unaffected by treatment and averaged 25.9 and 50.2 kg/d, respectively. Similarly, we observed no effect of treatment on yields of fat, protein, lactose, 3.5% fat-corrected milk, energy-corrected milk, feed efficiency, body weight, or body condition score. Milk fat concentration and yield were both reduced by the high RUFAL diets. We observed a tendency for AOX to increase the concentration of milk fat and decrease the concentration of milk protein. Yields of de novo and preformed fatty acids were not affected by treatment, although we detected a trend for a slight increase in the yield of 16-carbon fatty acid for AOX compared with CON. Treatment had only minor effects on individual milk fatty acids, except for the concentration and yield of linoleic acid, which were over 90% higher for AOX compared with CON. In conclusion, milk fat concentration and yield were reduced by a high RUFAL diet containing 15% distillers grains; however, supplementation with AOX did not overcome the MFD induced by this diet.     

Effect of dietary supplementation or cessation of magnesium-based alkalizers on milk fat output in dairy cows under milk fat depression conditions

We aimed to evaluate the effects of dietary supplementation with magnesium oxide and calcium-magnesium dolomite on milk fat synthesis and milk fatty acid profile or persistency in milk fat synthesis after their cessation in dairy cows under milk fat depression conditions. Twenty-four multiparous dairy cows in early lactation (mean ± standard deviation; 112 ± 14 d in milk) were used in a randomized complete block design. Milk fat depression was induced in all cows for 10 d by feeding a diet containing 35.2% starch, 28.7% neutral detergent fiber, and 4.8% total fatty acid (dry matter). The experiment was conducted in 2 periods. During the Mg-supplementation period (d 1–20), cows were randomly assigned to (1) the milk fat depression diet used during the induction phase (control; n = 8), (2) the control diet plus 0.4% magnesium oxide (MG; n = 8), or (3) the control diet plus 0.8% calcium-magnesium dolomite (CMC; n = 8). Compared with the control group, feeding the magnesium-supplemented diets increased milk fat concentration and yield by 12% within 4 d. During the 20-d Mg-supplementation period, both the MG and CMC diets increased milk fat concentration and yield, as well as 3.5% fat-corrected milk and energy-corrected milk yield, without affecting dry matter intake, milk yield, and milk protein and lactose concentrations. In the Mg-cessation period (d 21–30), all cows received the control diet, which resulted in a greater milk fat concentration and yield in the cows that had already received the MG and CMC diets in the Mg-supplementation period. Whereas, milk fat concentration and yield remained high after discontinuation of the magnesium-containing alkalizer until d 27. The difference in milk fat synthesis was associated with lower trans-10 C18:1 (?22%) and higher trans-11 C18:1 (+12.5%) concentrations in milk during the Mg-supplementation period. Furthermore, it was evident that within 2 d of supplementation, the trans-10:trans-11 ratio was lower in MG and CMC cows compared with cows receiving the control. This suggested that the effect of magnesium-based alkalizers on milk fat synthesis was mediated via a shift in ruminal biohydrogenation of cis-9,cis-12 C18:2 in the rumen. In conclusion, abrupt addition of magnesium oxide and calcium-magnesium dolomite increased milk fat synthesis, which persisted for 7 d after cessation of magnesium-based alkalizers. A similar ability to recover milk fat synthesis and normal fatty acid biohydrogenation pathways was observed for magnesium oxide and calcium-magnesium dolomite.     

Principal component and multivariate analysis of milk long-chain fatty acid composition during diet-induced milk fat depression

The objective of this study was to assess the relationship between individual milk fatty acids (FA) and diet-induced milk fat depression (MFD) using principal component analysis (PCA) and multivariate analysis (MA). Cow treatment observations (n = 63) from 3 published feeding experiments with lactating dairy cows were used in the analyses. In the PCA, principal component loading plots 1 (PC1) and 2 (PC2) described 55.9% of the total variation in milk FA and fat concentrations. Saturated FA (14:0, 16:0, and 17:0) and milk fat percentage showed negative loading for PC1. Trans-18:1 isomers (trans-6+7+8 to trans-15), trans-7, cis-9 conjugated linoleic acid (CLA), and trans-10, cis-12 CLA showed positive (opposite) loading, suggesting a negative relationship between these isomers and milk fat percentage. Cis-11, trans-13 CLA and cis-9, trans-11 CLA were associated with the PC2 axes (neutral), indicating that they were not associated with MFD. Multivariate analysis with milk fat percentage as the dependent variable and individual PC1 positive loading variables showed a breakpoint relationship for trans-6+7+8-, trans-9-, trans-10-, and trans-13+14-18:1 and a linear relationship for trans-11-, trans-12-, trans-15-18:1, trans-10, cis-12 CLA, and trans-7, cis-9 CLA. Subsequent MA was conducted on 41 treatment means from 12 independent experiments from the literature, in which concentrations of trans-6+7+8-, trans-9-, trans-10-, and trans-11-18:1, and cis-9 trans;-11, and trans-10, cis-12 CLA were reported. Significant negative effects of trans-9-18:1, trans-10-18:1, and trans-10, cis-12 CLA on milk fat percentage were observed. In this study, the PCA and MA showed that among trans-18:1 isomers, trans-10-18:1 was the most negatively correlated to milk fat percentage. However, the threshold concentration related to maximum MFD indicated that the relative potency was greatest for trans-6+7+8- and lowest for trans-10-18:1. These results suggested that trans-6+7+8-18:1 might be more important than trans-10-18:1 in MFD. Principal component analysis also showed that trans-10, cis-12 and trans-7, cis-9 CLA were the isomers most negatively correlated to milk fat percentage, implying a possible role of trans-7, cis-9 CLA in MFD. Additional experiments are needed to establish whether trans-7-18:1 is involved in MFD or that its effects are mediated via the endogenously synthesized trans-7, cis-9 CLA.     

Comparison of calcium salts and formaldehyde-protected conjugated linoleic Acid in inducing milk fat depression

Abomasal infusion studies have shown that trans-10, cis-12 conjugated linoleic acid (CLA) decreases milk fat synthesis. However, supplements of CLA must avoid rumen biohydrogenation for this technology to be applied to ruminants. Rumen protection methods would reduce CLA metabolism in the rumen and increase its supply to the small intestine. Our objective was to compare the efficacy of 2 forms of rumen-protected CLA at inducing milk fat depression. Three mid to late lactation Holstein cows each fitted with a rumen fistula were used in a 3 x 3 Latin square design. Treatments were: 1) control, 2) calcium salts of CLA (Ca-CLA), and 3) formaldehyde-protected CLA (FP-CLA). Supplements were designed to provide 10 g/d of trans-10, cis-12 CLA and were administered intraruminally once per day to ensure exact delivery of amount. Both CLA treatments substantially reduced milk fat yield and content compared with control, with the reductions in milk fat yield averaging 34% for the Ca-CLA treatment and 44% for the FP-CLA treatment. In contrast, milk yield, milk protein yield, and dry matter intake were unaltered by CLA treatment. Efficiency of transfer of trans-10, cis-12 CLA from the supplement into milk fat was 3.2 and 7.0% for Ca-CLA and FP-CLA, respectively. These values are much lower than transfer efficiencies reported for abomasally infused CLA, suggesting that much of the trans-10, cis-12 CLA present in the 2 formulations was biohydrogenated in the rumen. Overall, the extent of the reduction in milk fat yield indicates that both protection formulations are acceptable methods for the formulation of CLA supplements to induce milk fat depression in lactating dairy cows.     

Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates

Nine Holstein dairy cows were fed diets with increasing proportions of rapidly fermentable carbohydrates (RFCH) to investigate the effect on reticular pH, milk fat content (MFC), 18-carbon fatty acid proportions in blood plasma and milk, and bacterial community in buccal swab samples. Inter-animal variation was expected in terms of reticular pH response upon higher RFCH proportions, which would be reflected in the occurrence or not of milk fat depression (MFD). Moreover, this variation in occurrence of MFD was hypothesized to be related to differences in blood and milk fatty acid proportions and in the bacterial community in buccal samples. Cows were fed a total mixed ration throughout the experiment, which consisted of 4 periods: adaptation (d 0–4) and low (d 5–18), increasing (d 19–24), and high RFCH (d 25–28). During the increasing RFCH period, the standard concentrate (211 g of starch/kg of dry matter) was gradually and partly replaced by a concentrate high in RFCH (486 g of starch/kg of dry matter). The reticular pH was measured using a bolus and the time below pH 6.00 was calculated on a daily basis. On d 13, 14, 25, 27, and 28, plasma and milk samples were collected and analyzed for 18-carbon fatty acid proportions, and buccal swabs were collected for bacterial community analysis based on 16S rRNA gene amplicon sequencing. Inter-animal variation was observed in terms of reticular pH, which allowed us to divide the cows into 2 groups: tolerant (time below pH 6.00 ≤ 0.1 h/d) and susceptible cows (time below pH 6.00 ≥ 1.26 h/d). The lower reticular pH of susceptible cows was accompanied by lower MFC. Both groups already differed in reticular pH and MFC during the low-RFCH period. Furthermore, higher RFCH amounts did not decrease the reticular pH in either of the 2 groups. Nevertheless, MFD was observed in both groups during the high-RFCH period compared with the low-RFCH period. Lower MFC in animals with lower reticular pH or during the high-RFCH period was associated with a shift in 18-carbon fatty acids toward trans-10 at the expense of trans-11 intermediates, which was observed in plasma as well as in milk samples. Moreover, lower MFC was accompanied by shifts in the relative abundance of specific bacteria in buccal samples. Genera Dialister, Sharpea, Carnobacterium, Acidaminococcus, and uncultured genera belonging to the Betaproteobacteria were more abundant in situations with greater trans-10 proportions.     

Short communication: Effects of lysolecithin on milk fat synthesis and milk fatty acid profile of cows fed diets differing in fiber and unsaturated fatty acid concentration

Thirteen multiparous Holstein cows were used in a crossover design that tested the effect of lysolecithin in diets differing in neutral detergent fiber (NDF) and unsaturated fatty acid (FA) concentrations. Experimental periods were 20 d in length and included two 10-d phases. A standard fiber and lower fat diet was fed the first 10 d (30.5% NDF, no added oil, lower-risk phase) and a lower NDF and higher oil diet was fed during the second 10 d (29.0% NDF and 2% oil from whole soybeans and soybean oil, high-risk phase). Treatments were control and 10 g/d of lysolecithin (LYSO) extended in a ground corn carrier. Milk was sampled on d 0, 5, and 10 of each phase for determination of fat and protein concentration and FA profile. We found no effect of treatment or treatment by time interaction for dry matter intake, milk yield, or milk protein concentration. A treatment by time interaction was observed for milk fat concentration and yield. Milk fat concentration was higher in LYSO on d 5 of the lower-risk phase, but decreased progressively in both treatments during the high-risk phase. Milk fat yield was not different among treatments during the lower-risk phase, but was lower in LYSO on d 15 and tended to be lower on d 20 during the high-risk phase. Concentrations of milk de novo FA decreased and preformed FA increased during the high-risk phase, but we found no effect of treatment or treatment by time interactions. We noted an effect of time, but no treatment or treatment by time interactions for milk trans FA isomers. Briefly, trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid progressively decreased as trans-10 C18:1 and trans-10,cis-12 conjugated linoleic acid progressively increased during the high-risk phase. The LYSO increased milk fat concentration when feeding a higher fiber and lower unsaturated FA diet, but decreased milk fat yield when feeding a lower fiber and higher unsaturated FA diet, although biohydrogenation pathways and capacity did not appear to be modified. The effect of lysolecithin on rumen fermentation warrants further investigation, but is not recommended when feeding lower fiber and higher unsaturated fat diets.     

Effect of monensin on recovery from diet-induced milk fat depression

The objective of the present experiment was to investigate the effect of monensin (MN) on the time course of recovery from diet-induced milk fat depression. Milk fat depression was induced in all cows (n = 16) during the first phase of each period by feeding a low-fiber, high-unsaturated fat diet [25.3% neutral detergent fiber (NDF), 6.9% fatty acids (FA), and 3.24% C18:2] with MN (450 mg/cow per day) for 10 to 14 d. A recovery phase of 18 d followed, where cows were switched to a higher-fiber and lower unsaturated fat diet (31.2% NDF, 4.3% FA, and 1.7% C18:2). According to a crossover design, treatments during recovery were (1) control (no MN supplementation) or (2) continued MN supplementation. Milk yield, milk composition, and milk FA profile were measured every 3 d during recovery. No effect was observed of MN on dry matter intake or yield of milk, milk protein, and lactose. Milk fat concentration and yield increased progressively during recovery in both treatments. Monensin decreased milk fat yield from d 6 to 15, but it was the same as the control on d 18. A treatment by time interaction on milk fat concentration was detected, which was decreased by MN only on d 3 and 6. The yield of milk de novo synthesized FA increased progressively in both treatments and was not affected by treatment. Similarly, yield of 16-C FA increased progressively, but was decreased by MN on d 6 and 9. Preformed FA yield was lower in the MN group from d 6 to 15, but was not different from the control on d 18. Importantly, milk FA concentration of trans-10 C18:1 and trans-10,cis-12 conjugated linoleic acid rapidly decreased in both groups; however, MN slightly increased trans-10 C18:1 concentration above baseline on d 15 and 18. In conclusion, MN supplementation had minimal effect on recovery of normal rumen biohydrogenation and de novo FA synthesis during recovery from milk fat depression by correction of dietary starch, NDF, and polyunsaturated FA concentration, but moderately decreased recovery of preformed FA in milk.     

Interaction of molasses and monensin in alfalfa hay- or corn silage-based diets on rumen fermentation, total tract digestibility, and milk production by Holstein cows

Sugar supplementation can stimulate rumen microbial growth and possibly fiber digestibility; however, excess ruminal carbohydrate availability relative to rumen-degradable protein (RDP) can promote energy spilling by microbes, decrease rumen pH, or depress fiber digestibility. Both RDP supply and rumen pH might be altered by forage source and monensin. Therefore, the objective of this study was to evaluate interactions of a sugar source (molasses) with monensin and 2 forage sources on rumen fermentation, total tract digestibility, and production and fatty acid composition of milk. Seven ruminally cannulated lactating Holstein cows were used in a 5 × 7 incomplete Latin square design with five 28-d periods. Four corn silage diets consisted of 1) control (C), 2) 2.6% molasses (M), 3) 2.6% molasses plus 0.45% urea (MU), or 4) 2.6% molasses plus 0.45% urea plus monensin sodium (Rumensin, at the intermediate dosage from the label, 16 g/909 kg of dry matter; MUR). Three chopped alfalfa hay diets consisted of 1) control (C), 2) 2.6% molasses (M), or 3) 2.6% molasses plus Rumensin (MR). Urea was added to corn silage diets to provide RDP comparable to alfalfa hay diets with no urea. Corn silage C and M diets were balanced to have 16.2% crude protein; and the remaining diets, 17.2% crude protein. Dry matter intake was not affected by treatment, but there was a trend for lower milk production in alfalfa hay diets compared with corn silage diets. Despite increased total volatile fatty acid and acetate concentrations in the rumen, total tract organic matter digestibility was lower for alfalfa hay-fed cows. Rumensin did not affect volatile fatty acid concentrations but decreased milk fat from 3.22 to 2.72% in corn silage diets but less in alfalfa hay diets. Medium-chain milk fatty acids (% of total fat) were lower for alfalfa hay compared with corn silage diets, and short-chain milk fatty acids tended to decrease when Rumensin was added. In whole rumen contents, concentrations of trans-10, cis-12 C_18:2 were increased when cows were fed corn silage diets. Rumensin had no effect on conjugated linoleic acid isomers in either milk or rumen contents but tended to increase the concentration of trans-10 C_18:1 in rumen samples. Molasses with urea increased ruminal NH₃-N and milk urea N when cows were fed corn silage diets (6.8 vs. 11.3 and 7.6 vs. 12.0 mg/dL for M vs. MU, respectively). Based on ruminal fermentation characteristics and fatty acid isomers in milk, molasses did not appear to promote ruminal acidosis or milk fat depression. However, combinations of Rumensin with corn silage-based diets already containing molasses and with a relatively high nonfiber carbohydrate:forage neutral detergent fiber ratio influenced biohydrogenation characteristics that are indicators of increased risk for milk fat depression.     

Estimation of genetic parameters for milk fat depression in dairy cattle

The objective of this study was to apply reaction norm models to milk recording data to investigate genetic variation in and environmental sensitivity of susceptibility to milk fat depression (MFD). Data comprised 556,276 test-day records of 80,493 heifers in 1043 herds. Breeding values and genetic variances for fat percentage and fat yield were estimated by applying random regression models to average herd-test-day fat percentage. Genetic and permanent environmental correlations between fat yield expressed in different environments ranged, respectively, from 0.83 to 1.00 and from 0.29 to 1.00. Genetic and permanent environmental correlations between fat percentage expressed in different environments ranged, respectively, from 0.87 to 1.00 and from -0.05 to 0.99. Two traits were defined for MFD. The first trait reflected variation of milk fat percentage of animals within lactation after correction for year-season, herd-test-day, age-at-calving, and stage-of-lactation. This trait had an estimated heritability of about 5% and a genetic correlation between the fifth and 95th percentile of the data of 0.50. The second trait reflected the deviation of an animal's fat percentage on a test-day from its expected fat percentage based on fat percentage on the first test-day. This trait had an estimated heritability of about 4% and a genetic correlation between the fifth and 95th percentile of the data of 0.43. The correlation between estimated breeding values of sires for the 2 MFD traits was -0.3. Our results suggest that genetic variation in susceptibility to MFD is present and that selection for reduced susceptibility to MFD is possible.     

Short communication: Regulation of milk fat yield and fatty acid composition by insulin

Diet-induced milk fat depression in dairy cows has been known for many years and several theories have been proposed. One that continues to receive support is the glucogenic-insulin theory. Previous studies testing this theory using a hyperinsulinemic-euglycemic clamp have had variable results attributable to variability in the use of body fat reserves as a source of milk fatty acids. Our objective was to test the glucogenic-insulin theory using cows immediately postpartum, a period when the use of body fat for milk fat synthesis is greatest. During wk 2 postpartum, 5 cows were given a 2-d baseline period and then clamped for 4 d. Insulin was increased more than 2-fold during the clamp while the blood glucose concentration was maintained. Milk yield was not altered by administration of the clamp (38.7 vs. 39.0 ± 1.4 kg/d); however, the milk fat percentage and yield were reduced by 27% and plasma nonesterified fatty acids were reduced by 68%. Analysis of the milk fatty acid composition revealed that the decrease in milk fat yield during use of the clamp was almost exclusively due to reductions in preformed fatty acids; this is the exact opposite of what is observed with diet-induced milk fat depression. Therefore, our results do not support the glucogenic-insulin theory of diet-induced milk fat depression. The results further indicated that reductions in milk fat observed previously with hyperinsulinemic-euglycemic clamps or with glucose or propionate infusions were most likely consequences of the ability of insulin to inhibit lipolysis, thereby limiting the mammary availability of preformed fatty acids mobilized from body reserves.     

Effects of intravenous infusion of conjugated diene 18:3 isomers on milk fat synthesis in lactating dairy cows

It has been previously established that trans-10, cis-12 conjugated linoleic acid plays an important role in milk fat depression (MFD). However, in many situations of dietary induced MFD, the reduction in milk fat synthesis is much greater than what would be predicted based on the milk fat concentration of trans-10, cis-12 18:2. These observations suggest that other biohydrogenation intermediates could be implicated in MFD. The objective of this study was to evaluate the effects on milk fat synthesis of an intravenous administration of 2 conjugated diene 18:3 isomers (cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15 18:3), which are intermediates in ruminal biohydrogenation of α-linolenic acid. Three multiparous Holstein dairy cows (days in milk = 189 ± 37 d; body weight = 640 ± 69 kg; mean ± standard deviation), fitted with indwelling jugular catheters, were randomly assigned to a 3 × 3 Latin square design. For the first 5 d of each period, cows were infused intravenously with a 15% lipid emulsion providing 1) cis-9, trans-11, cis-15 18:3 + cis-9, trans-13, cis-15 18:3 + trans-10, cis-12 18:2 (CD18:3 + CLA); 2) cis-9, cis-12, cis-15 18:3 + cis-9, cis-12 18:2 as a control (ALA + LA); or 3) cis-9, cis-12, cis-15 18:3 + trans-10, cis-12 18:2, as a positive control (ALA + CLA). Milk production was recorded, and milk was sampled daily at each milking for analyses of fat, protein, lactose, milk urea nitrogen, and somatic cell count. Dry matter intake, milk yield, and milk protein were not affected by treatment. Over the experimental period, milk fat content was decreased by 7% for cows that received either ALA + CLA or CD18:3 + CLA compared with ALA + LA. The temporal pattern of milk fat content showed a linear decrease during the infusion period for ALA + CLA and CD18:3 + CLA treatment groups. The transfer efficiencies of conjugated diene 18:3 isomers into milk fat averaged 39 and 32% for cis-9, trans-11, cis-15 18:3 and cis-9, trans-13, cis-15 18:3, respectively. The CD18:3 + CLA treatment had no effect on milk fat concentration beyond that attributable to its trans-10, cis-12 18:2 content. In conclusion, results from the current study offered no support for a role of either cis-9, trans-11, cis-15 18:3 or cis-9, trans-13, cis-15 in MFD.