Background: The presence and concentration of lipids in serum of dairy cows have significant implications for both animal health and productivity and are potential biomarkers for several common diseases. However, information on serum lipid composition is rather fragmented, and lipid remodelling during the transition period is only partially understood. Methods: Using a combination of reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS), hydrophilic interaction-mass spectrometry (HILIC-MS), and lipid annotation software, we performed a comprehensive identification and quantification of serum of dairy cows in pasture-based Holstein-Friesian cows. The lipid remodelling induced by negative energy balance was investigated by comparing the levels of all identified lipids between the fresh lactation (5–14 days in milk, DIM) and full lactation (65–80 DIM) stages. Results: We identified 535 lipid molecular species belonging to 19 classes. The most abundant lipid class was cholesteryl ester (CE), followed by phosphatidylcholine (PC), sphingomyelin (SM), and free fatty acid (FFA), whereas the least abundant lipids included phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), acylcarnitine (AcylCar), ceramide (Cer), glucosylceramide (GluCer), and lactosylceramide (LacCer). Conclusions: A remarkable increase in most lipids and a dramatic decrease in FFAs, AcylCar, and DHA-containing species were observed at the full lactation compared to fresh lactation stage. Several serum lipid biomarkers for detecting negative energy balance in cows were also identified.

Context: On pasture-based dairy farms, herd behavioural dynamics result in a milking order that remains consistent over time. Previous research has demonstrated that cows that are consistently early in the milking order routinely access pasture of greater biomass and nutrient density than do later-milked cows, because they access the paddock first. This effect is likely to explain why early cows commonly produce more milk than do later cows. We hypothesised that in commercial dairy herds, milk yield would decline as milking order progressed, and there would be a reduction in pasture mass and nutritive value before the last cows returned to the paddock. Aims: This study aimed to validate observations previously demonstrated under controlled experimental conditions, in commercial dairy herds in Gippsland, Victoria. Methods: This research was repeated across three commercial farms, with four grazing sessions observed per farm. Cows were milked twice daily, with daily individual milk yields and milking times obtained. Pasture measurements were collected pre-grazing, hourly for the first 3 h of grazing after the morning milking (while cows progressively entered the paddock), and post-grazing after the cows exited the paddock. At each sampling, pasture mass was estimated using a calibrated rising-plate metre, and pasture samples were collected for analyses of nutritive characteristics. Key results: Milking order was consistent, and the last cows produced on average 4.7 L/cow.day less milk than did the first cows. As grazing progressed, pasture mass, crude protein concentrations and metabolisable energy declined and fibre concentrations increased. Conclusions: This study demonstrated a decrease in pasture mass and nutrient density as grazing progressed. The earliest cows had access to more pasture of higher nutrient density and produced more milk than did later-milked cows. Implications: This study validated that the milk yield of cows in commercial herds is influenced by milking order, which is likely to be mediated at least in part by the quantity and nutrient density on offer to earlier versus later-milked cows. Other factors may also influence milking order and milk yield, but these factors were not accounted for in this research. Future research should focus on mitigation strategies to negate this effect by improving nutrient intake of later-milked cows.

Both in vitro and in vivo studies have shown the potential of Lactobacillus spp. to mitigate enteric methane. However, their mitigation impact on early-lactation dairy cows grazing high-pasture diets has not been studied. This study evaluated the effect of supplementing freeze-dried or liquid Lactobacillus spp. products on enteric methane emission from early lactation dairy cows offered a high forage diet. Forty spring calving Holstein Friesian dairy cows, grazing a perennial ryegrass (PRG) (Lolium perenne L.) based pasture, milked twice daily, were randomly allocated to one of the 3 treatment diets offered for 35 d: 1) CON: Control, fed a basal diet (n = 14); 2) FLA: basal diet plus freeze-dried Lactobacillus spp. (n = 13) delivering 5.0 × 10¹⁰ cfu/milking, and 3) LLA: basal diet plus Liquid Lactobacillus spp. (n = 13) delivering 5.75 × 10¹⁰ cfu/milking. The basal diet comprised 6.9 kg DM/day of a grain mix plus PRG pasture at an allowance of approximately 25 kg DM/cow per day. Covariate observations were made for the first 5 d of the 40-d experiment, before commencing treatments. During the last 5 d of the experiment, individual forage dry matter intake for each cow was estimated using the n-alkane technique, and methane emissions from individual cows were measured using the modified sulfur hexafluoride (SF₆) tracer technique. Data were analyzed using a completely randomized design based ANCOVA. Methane production (g/day), yield (g/kg DMI) and intensity (g/kg energy corrected milk) were not affected by the inclusion of either form of Lactobacillus spp. in the diet, although a numerical reduction of 6% in methane yield was observed in the FLA group, compared with the control group. Methane emissions from the CON cows were approximately 38% lower than those estimated using inventory factors. This is likely due to the low fiber concentration and low DM concentration of the PRG offered likely resulting in a high ruminal passage rate, reducing ruminal retention time and extent of fermentation. The high passage rate could have also restricted the colonization potential of supplemented Lactobacillus spp. This, combined with the large rumen volume of dairy cows and low dose rate may in part explain the limited methane mitigation. Notably, production responses to Lactobacillus spp. supplementation have been more evident in small ruminants, suggesting species-specific differences. These findings highlight the need for tailored probiotic strategies, particularly in pasture based dairy systems.

Background: Negative energy balance (NEB) during the transition period is associated with profound changes in the body condition and metabolic dynamics of dairy cows. However, the detailed lipidomic changes in milk induced by NEB are unclear, and lipid biomarkers that indicate the energy status of cows remain to be established. Methods: Using a combination of GC-FID, HILIC-MS and RP-LC-MS, we performed a systematic comparison of lipid composition between early lactating (DIM: 5–14) and mid-lactating (DIM: 65–80) milk. Results: We found that NEB in cows caused a profound modification in the profile of all the lipid classes surveyed, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM), lysophosphatidylcholine (LPC), PC-plasmalogen (PCP), PE-plasmalogen (PEP), lactosylceramide (LacCer), acylcarnitine (AcylCar) and triglycerides (TAGs). Except for LPC and AcylCar, which were reduced and increased, respectively, by NEB, the responses of other lipid classes varied across different species. For phospholipids and TAGs, species containing de novo FAs (C4:0–C16:0) and odd-chain FAs (C15:0 and C17:0) were markedly downregulated, whereas those comprising long-chain preformed FAs were upregulated by NEB. Conclusions: Comprehensive lipidomic profiling of early and mid-lactating milk from two large cohorts of cows allowed us to identify nine lipids (PE 33:1, LacCer 32:1, LacCer 39:1, LacCer 41:1, SM 36:1, SM 36:2, SM 37:1, PEP 38:4 and PEP 38:5) as potential biomarkers of NEB in dairy cows.