Heat stress during the dry period negatively affects hepatic metabolism and cellular immune function during the transition period, and milk production in the subsequent lactation. However, the cellular mechanisms involved in the depressed mammary gland function remain unknown. The objective of the present study was to determine the effect of heat stress during the dry period on various indices of mammary gland development of multiparous cows. Cows were dried off approximately 46 d before expected calving and randomly assigned to 2 treatments, heat stress (HT, n=15) or cooling (CL, n=14), based on mature equivalent milk production. Cows in the CL treatment were provided with sprinklers and fans that came on when ambient temperatures reached 21.1°C, whereas HT cows were housed in the same barn without fans and sprinklers. After parturition, all cows were housed in a freestall barn with cooling. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rates recorded at 1500 h on a Monday-Wednesday-Friday schedule from dry off to calving. Milk yield and composition were recorded daily up to 280 d in milk. Daily dry matter intake was measured from dry off to 42 d relative to calving. Mammary biopsies were collected at dry off, -20, 2, and 20 d relative to calving from a subset of cows (HT, n=7; CL, n=7). Labeling with Ki67 antigen and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling were used to evaluate mammary cell proliferation and apoptosis, respectively. The average temperature-humidity index during the dry period was 76.6 and not different between treatments. Heat-stressed cows had higher rectal temperatures in the morning (38.8 vs. 38.6°C) and afternoon (39.4 vs. 39.0°C), greater respiration rates (78.4 vs. 45.6 breath/min), and decreased dry matter intake (8.9 vs. 10.6 kg/d) when dry compared with CL cows. Relative to HT cows, CL cows had greater milk production (28.9 vs. 33.9 kg/d), lower milk protein concentration (3.01 vs. 2.87%), and tended to have lower somatic cell score (3.35 vs. 2.94) through 280 d in milk. Heat stress during the dry period decreased mammary cell proliferation rate (1.0 vs. 3.3%) at -20 d relative to calving compared with CL cows. Mammary cell apoptosis was not affected by prepartum heat stress. We conclude that heat stress during the dry period compromises mammary gland development before parturition, which decreases milk yield in the next lactation.

Dietary sources of fatty acids were evaluated for their influence on oocyte quality and follicular development using 54 lactating cows in summer. Fat supplements were 1) sunflower oil (80% cis 18:1), 2) Ca salt of transoctadecenoic acids (57% trans 18:1), 3) Ca salt of vegetable oils (30% 18:2), and 4) linseed oil (56% 18:3 and 16% 18:2). Fats were fed at 1.35% of dietary dry matter beginning at 5 wk prior to expected calving date and at 1.5% (oils) and 1.75% (Ca salts) of dietary dry matter for 15 wk after parturition. Four days following a programmed induced ovulation, 5 transvaginal oocyte aspirations were performed 3 or 4 d apart. Three days after the last aspiration, PGF2alpha was injected, followed 3 d later by a GnRH injection and a timed artificial insemination (d 0) 16 to 20 h later. For the first 4 aspirations, oocytes grading 1 or 2 were used for in vitro embryo production. Total cell number and the proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive blastomeres were analyzed at d 8. At the fifth aspiration, the occurrence of metaphase II, group II caspase activity, and TUNEL labeling were determined after oocyte maturation. A total of 1,011 oocytes were collected. The proportion of oocytes with high caspase activity was greater for grade 3 compared with grades 1 and 2 (37.5 vs. 1.54 and 1.61%). Feeding polyunsaturated fatty acids, as compared with monosaturated fatty acids, failed to affect oocyte quality, as demonstrated by subsequent embryo development. Cows fed 18:2- or 18:3-enriched diets had a larger preovulatory follicle at insemination and subsequent volume of the corpus luteum compared with those fed cis 18:1 or trans 18:1 diets (16.8, 16.2 vs. 15.0, 14.9 +/- 0.7 mm; 7,323, 8,208 vs. 6,033, 5,495 +/- 644 mm3, respectively). The previously documented benefits of polyunsaturated fatty acids on reproductive performance appear to reflect actions at alternative biological windows in lactating dairy cows.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein–protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Environmental factors, especially temperature and light exposure, influence the health and productivity of dairy cows during lactation, possibly via similar physiological mechanisms. For example, heat stress is a critical component of decreased milk yield during summer. However, less is known about the effect of heat stress during the dry period. The objective of this study was to evaluate the effects of heat stress prepartum under a controlled photoperiod on lactation performance and hepatic metabolic gene expression of periparturient multiparous Holstein cows (n = 16). Cows were dried off approximately 46 d before expected calving date and assigned to treatment randomly after blocking by mature equivalent milk production and parity. Treatments consisted of either heat stress (HT) or cooling (CL) with fans and sprinklers, both under a photoperiod of 14L:10D. Rectal temperature was measured twice daily during the dry period. After calving, cows were housed in a freestall barn with cooling devices, and milk yield was recorded daily up to 210 d in milk. Blood samples were taken from dry off until +42 d relative to calving for metabolites and from -2 until +2 d relative to calving for hormone analysis. Daily dry matter intake was measured from -35 to +42 d relative to calving. Liver biopsies were collected at dry off, -20, +2, and +20 d relative to calving for cows on HT (n = 5) and CL (n = 4) to measure mRNA expression of suppressors of cytokine signaling-2 (SOCS-2), insulin-like growth factor binding protein-5 (IGFBP-5), a key transcription factor in lipid biosynthesis (SREBP-1c), and enzymes of lipid metabolism (FASN, ACACA, and ACADVL) by real-time quantitative PCR. Heat stress increased rectal temperatures (39.2 vs. 38.8 degrees C), plasma prolactin concentrations at -1 (171 vs. 79 ng/mL) and 0 d (210 vs. 115 ng/mL) relative to calving, and decreased dry matter intake at 0 and +14 d relative to calving and 3.5% fat-corrected milk postpartum (26.1 vs. 35.4 kg/d) compared with CL cows. Relative to CL cows, hepatic mRNA expression of SOCS-2 and IGFBP-5 was downregulated in HT cows. Expression of ACADVL was upregulated in CL cows at d +2 but downregulated at d +20 relative to HT cows. Concentrations of C16:0 and cis C18:1 were greater in the milk and liver of CL cows compared with HT cows, which reflects greater lipid mobilization. These results suggest that heat-stress abatement in the dry period improves subsequent lactation, possibly via suppression of plasma prolactin surge around calving, SOCS-2 expression, and regulation of hepatic lipid metabolism.

Heat stress (HT) and photoperiod affect milk production and immune status of dairy cows. The objective was to evaluate the effects of HT abatement prepartum under controlled photoperiod on hepatic metabolic gene expression and cellular immune function of periparturient Holstein cows (n=21). Cows were dried off 46 d before expected calving date and assigned to treatments by mature equivalent milk production. The treatments were 1) HT and 2) cooling (CL), both imposed during a photoperiod of 14L:10D. Rectal temperature was measured twice daily, whereas respiration rate was measured 3 times/wk at 1500 h during the entire dry period. After calving, cows were housed in a freestall barn with cooling, and milk yield was recorded daily up to 140 d in milk. Liver samples were taken at dry off, -20, 2, and 20 d relative to calving by biopsy. Under a similar schedule, neutrophil function was determined in blood of cows on HT (n=12) and CL (n=9). Blood samples were taken on -46, -32, -18, 0, 14, 28, and 42 d relative to calving for measurement of metabolites and were collected twice daily from -7 to 2 d relative to calving for prolactin (PRL) analysis. The HT cows had greater concentrations of PRL at 0 d relative to calving (150 vs. 93; SEM=11 ng/mL) and had higher afternoon rectal temperatures (39.4 vs. 39.0; SEM=0.04°C) and elevated respiration rates (78 vs. 56; SEM=2 breaths/min) during the prepartum period compared with CL cows. Relative to HT cows, CL cows had greater hepatic expression of PRL-R, SOCS-3, and CAV-1 mRNA. Neutrophil oxidative burst was greater in CL cows relative to HT cows at 2 d (61 vs. 42; SEM=6%) and at 20 d (62 vs. 49; SEM=5%) relative to calving, and phagocytosis was greater in CL cows at 20 d (47 vs. 33; SEM=4%) relative to calving compared with HT cows. Humoral response, as measured by IgG secretion against ovalbumin challenge, was greater for CL cows at -32 d (0.44 vs. 0.33; SEM=0.05 OD) and -21 d (0.60 vs. 0.50±0.04 OD) relative to calving compared with HT cows. These results suggest that HT abatement during the dry period improved innate and acquired immune status as measured by neutrophil function and immunoglobulin secretion against ovalbumin challenge, and altered hepatic gene expression related to PRL signaling in the periparturient period or subsequent lactation.

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Heat stress negatively affects cow performance, compromises immune function, and increases susceptibility to metabolic disorders, particularly during the dry period and as cows transition from gestation to lactation. Metabolic adaptations of the liver are critical for successful transition, yet it is unclear how heat stress affects metabolic pathways within the liver at the proteomic level. The objective of this study was to investigate the liver proteome of postpartum cows that were cooled or heat stressed during the dry period to gain insight into how protein expression is altered by prior heat stress and may contribute to performance and disease outcomes. During the dry period, cows were either housed in shaded barns with fans and water soakers [cooled group (CL); n = 5] or in shaded barns lacking these cooling devices [heat-stressed group (HT); n = 5]. Liver biopsies were collected at 2 d postpartum, and protein content was analyzed by label-free quantitative shotgun proteomics (nanoscale liquid chromatography coupled to tandem mass spectrometry). In the most comprehensive bovine liver proteomics analysis completed to date, we identified 3,270 proteins, 75 of which were differentially expressed between HT and CL cows (fold change ±1.2). The top pathways differing between HT and CL cows were oxidative phosphorylation, mitochondrial dysfunction, farnesoid X receptor/retinoid X receptor (FXR/RXR) activation, and the methylmalonyl pathway. Cooling cows during the dry period likely improves ATP production, reduces oxidative stress, and prevents excessive accumulation of hepatic triglycerides and cholesterol, which may contribute to greater milk yield and lower susceptibility to transition-related diseases.

The main objective of this study was to determine the association of dry matter intake as percentage of body weight (DMI%BW) and energy balance (EB) prepartum (−21 d relative to parturition) and postpartum (28 d) with calving disorders (CDZ; dystocia, twins, and stillbirths; n = 101) and metritis (n = 114). For this, DMI%BW and EB were the independent variables and CDZ and metritis were the dependent variables. A secondary objective was to evaluate prepartum DMI%BW and EB as predictors of CDZ and metritis. For this, CDZ and metritis were the independent variables and DMI%BW and EB were the dependent variables. Data from 476 cows from 9 experiments were compiled. Cows that developed CDZ had lesser postpartum DMI%BW from d 3 to 12 and lesser energycorrected milk (ECM) than cows that did not develop CDZ. Dry matter intake as percentage of BW and EB prepartum did not affect the odds of CDZ. Cows with metritis had lesser prepartum DMI%BW and EB. Each 0.1-percentage point decrease in the average DMI%BW and each 1-Mcal decrease in the average EB in the last 3 d prepartum increased the odds of having metritis by 8%. The average DMI%BW and EB during the last 3 d prepartum produced significant cut-offs to predict metritis postpartum, which were ≤1.6%/d and ≤2.5 Mcal/d, respectively. Cows that developed metritis had lesser overall postpartum DMI%BW and ECM and lesser EB from d 2 to 5 and from d 7 to 11 than cows that did not develop metritis. The main limitation in this study is that the time-order of disease relative to DMI%BW and ECM is inconsistent such that postpartum outcomes were measured before and after disease, which was diagnosed at variable intervals after calving. In summary, prepartum DMI%BW and EB were associated with and were predictors of metritis although the effect sizes were small for metritis, and calving disorders and metritis were associated with decreased DMI%BW and ECM postpartum.