Endometrial responsiveness to interferon-tau and its association with subsequent reproductive performance in dairy heifers.

Our objectives were to evaluate the endometrial responsiveness of dairy heifers to an intrauterine infusion of recombinant bovine interferon-tau (rbIFN-τ) and to associate endometrial responses to rbIFN-τ with subsequent reproductive performance. In Experiments 1 and 2, cyclic heifers were enrolled in a 5-d CIDR Cosynch program for estrous synchronization, and blood sampling and ultrasonography examinations were performed on d 0, 4, 7, 11, and 14 of the estrous cycle. In Experiment 1, heifers were randomly assigned to receive an intrauterine infusion containing 2 µg of rbIFN-τ (rbIFN-τ = 19) or saline (CTRL = 19) into the uterine horn ipsilateral to the corpus luteum (CL) on d 14 of the estrous cycle. Six hours after the infusion, the infused uterine horn was flushed for sampling of the uterine luminal fluid (ULF) for analyses of composition, and the endometrium was biopsied for transcriptomics. In Experiment 2, 100 heifers received an intrauterine infusion of rbIFN-τ, and the same procedures for uterine sample collection described in Experiment 1 were performed. After the intrauterine test, heifers were enrolled in a breeding program and classified as highly fertile (HF; pregnant at first AI) or subfertile (SF; not pregnant at first AI). Statistical analyses were performed using linear regression models, which included the effects of treatment (Experiment 1: CTRL vs. rbIFN-τ) or fertility group (Experiment 2: HF vs. SF) and block of samples. Intrauterine infusion of rbIFN-τ increased the expression of classical interferon-stimulated genes in the endometrium (e.g., ISG15, MX1, OAS2, IRF9, and USP18), and an antiviral response was predicted to be the main downstream effect of the transcriptome changes. In addition, rbIFN-τ increased the abundance of cholesterol, glycerol, and the overall concentration of oxylipins in the ULF. Analysis of endometrial transcriptome between HF and SF heifers revealed important differences in the expression of proteins associated with cell signaling, metabolism, attachment, and migration, with a large representation of genes encoding extracellular matrix proteins. In general, differently expressed genes were expected to be downregulated by IFN-τ but seemed to fail to be downregulated in SF heifers, resulting in higher expression in SF compared with HF heifers. Subfertile heifers had lower concentrations of glycerol and an altered profile of oxylipins in the ULF, with lower abundance of oxylipins derived from arachidonic acid and dihomo-γ-linolenic acid, and greater abundance of oxylipins derived from linoleic acid. Measurements of ovarian function did not differ between groups and, therefore, did not influence the observed results in uterine biology. In conclusion, the endometrial responsiveness to IFN-τ is variable among individuals and associated with subsequent fertility of heifers, indicating that communication between conceptus and endometrium is critical for the uterine receptivity and survival of pregnancy.

The effect of supplementary selenium source on apparent and true absorption, retention, performance, and selenium status in lactating Holstein cows.

Selenium is an essential trace mineral for dairy cattle and can be provided in the diet in various forms that may differ in bioavailability. The objective of this study was to determine how source of Se affects animal performance, Se status, retention, and apparent and true absorption. Multiparous Holstein cows (n = 24; 597 ± 49 kg body weight) were blocked by days in milk (DIM; 161 ± 18) and randomly assigned to receive 0.3 mg Se/kg of dry matter (100% of NASEM requirements) of either organic Se (ORG; selenized yeast) or inorganic Se (INO; sodium selenite). The Se premix was top-dressed on a common total mixed ration fed daily and mixed into the top 15 cm directly before feeding. Following an 11-wk adaptation period, cows received simultaneous infusions of an intraruminal isotope dose of 77Se in the same chemical form as the premix, and an intravenous dose of 82Se in an inorganic form. Infusions were followed by a 4-d period of blood and rumen fluid sampling, and total collection of feces, urine, and milk. Daily dry matter intake (23 ± 0.6 kg), milk yield (35 ± 1.2 kg), and serum Se (0.11 ± 0.003 µg/g) were not different between treatments during the adaptation period, but milk Se concentrations were greater for ORG compared with INO. Serum 77Se maximum concentration (Cmax) and area under the curve (AUC) were not different between treatments for 72 h following infusion, but rumen fluid 77Se AUC was higher for ORG than INO. Apparent absorption (64 ± 1.4%), and retention (44 ± 1.5%) of the 77Se dose did not differ between treatments. True absorption was calculated using 82Se enrichment in serum and feces and was determined to be 69 ± 1.3% and did not differ between treatments. Fecal excretion of the 77Se dose was not different between treatments (36 ± 1.4%), but ORG had lower urinary excretion and higher milk excretion compared with INO. These results indicate that organic Se resulted in greater Se concentration of milk and lower urinary Se excretion into the environment, but absorption, Se status, and performance of the cow were not affected by Se source at this supplementation level.

Associations of postpartum health with progesterone after insemination and endocrine signaling during early pregnancy in dairy cows.

Our objective was to investigate associations of postpartum health with serum progesterone (P4) concentrations after first artificial insemination (AI), expression of interferon-stimulated genes (ISG) in blood leukocytes, and serum pregnancy-associated glycoprotein (PAG) during early pregnancy in dairy cows. In this observational study, Holstein cows (n = 475) from 2 commercial herds were enrolled 3 wk before expected parturition. Body condition score (BCS) and lameness were assessed at enrollment, and total Ca, haptoglobin, and nonesterified fatty acids (NEFA) were measured in serum at 2 and 6 ± 2 d in milk (DIM). Blood ß-hydroxybutyrate and metritis were evaluated at 4, 8, 11, and 15 ± 2 DIM, and purulent vaginal discharge (PVD) and endometritis based on endometrial cytology were diagnosed at 35 DIM. Onset of cyclicity was assessed by biweekly P4 measurements. The BCS was assessed at enrollment and at 63 DIM, and lameness at enrollment and at 21 and 49 DIM. First AI was based on estrus detection until ∼75 DIM, or synchronization for timed AI thereafter. Serum P4 was measured at d 8 and 12 after first AI; expression of ISG (ISG15 and RTP4) in blood leukocytes was assessed at d 19; and PAG in serum of pregnant cows was measured at d 29, 33, and 40. Multivariable linear regression models were built including health variables (with metabolites categorized via receiver operating characteristic curve analysis) and covariates (season, milk yield, AI method, DIM), accounting for pregnancy status (for ISG and PAG models), repeated measures (for P4 and PAG models), and herd as random. Results are reported comparing cows affected versus unaffected by each predictor. Serum P4 concentrations were lesser at d 8 after AI in cows that had total Ca ≤2.09 mM (3.6 vs. 4.0 ± 0.2 ng/mL) at 2 DIM or a clinical disease (3.7 vs. 4.0 ± 0.2 mg/mL), and P4 tended to be lesser at d 8 in cows that had PVD (3.6 vs. 4.0 ± 0.2 ng/mL) or any uterine disease (3.7 vs. 4.1 ± 0.2 ng/mL). Among cows pregnant at d 29, relative ISG15 expression (fold change) at d 19 was greater in cows that had lameness (5.10 [4.18-6.18] vs. 3.14 [3.10-3.19]) but tended to be lesser in cows that had endometritis (3.20 [2.89-3.54] vs. 4.29 [4.20-4.35]), and relative expression of RTP4 tended to be lesser in cows that had displaced abomasum (1.02 [0.62-1.67] vs. 1.75 [1.73-1.78]). Serum PAG was lesser at d 29 (3.8 vs. 4.2 ± 0.2 sample-to-positive ratio [S:P]) in cows that had NEFA ≥0.73 mM at 6 DIM, and lesser at d 33 (3.9 vs. 4.5 ± 0.2 S:P) and 40 (3.1 vs. 3.7 ± 0.2 S:P) in cows that had prepartum BCS ≥3.75. These findings suggest long-term effects of metabolic or reproductive tract disorders on luteal function after first AI, pregnancy recognition signaling, and placental function during early pregnancy. These associations may partially explain some of the pathways linking postpartum health and fertility in dairy cows.

Associations of inflammatory and reproductive tract disorders postpartum with pregnancy and early pregnancy loss in dairy cows.

Our objective was to describe associations of postpartum health with pregnancy and pregnancy loss (P-LOSS) from d 19 to 40 after first postpartum artificial insemination (AI) in lactating Holstein cows. In 2 commercial dairy herds in Ontario, Canada, 468 Holstein cows were enrolled 21 ± 3 d before expected parturition when body condition score (BCS) and lameness were assessed. Serum total Ca, haptoglobin (Hp), and nonesterified fatty acids (NEFA) were measured at 2 and 6 ± 2 d in milk (DIM). Blood ß-hydroxybutyrate (BHB) measurement and metritis detection were done at 4, 8, 11, and 15 ± 2 DIM. Cows were examined for endometritis (ENDO; ≥11.5% polymorphonuclear cells in endometrial cytology) and purulent vaginal discharge (PVD) at 35 ± 3 DIM. Lameness was assessed again at 21 and 49 ± 3 DIM and BCS at 63 ± 3 DIM. First postpartum AI occurred primarily (86%) based on detection of estrus by activity monitors, on average (± standard deviation) at 65 ± 9 DIM, and the remaining cows received timed AI at 86 ± 18 DIM. Serum progesterone (P4) was measured on d 8 and 12 after AI, and pregnancy at first AI (P/AI) was estimated by the expression of ISG15 in peripheral blood leukocytes at d 19 after AI and by pregnancy-associated glycoprotein in serum at d 29, 33, and 40 after AI. Each metabolite (Ca, Hp, NEFA, and BHB) was categorized above or below a cut-point identified with receiver operating characteristic curve analysis associated with P/AI confirmed by ultrasound at d 33 from a larger data set. Data were analyzed using multivariable mixed logistic regression models, accounting for parity, health variables, covariates (season at calving and at AI, milk yield at first Dairy Herd Improvement Association test [categorized into terciles], AI method, and DIM), and herd. The proportions of cows classified pregnant at d 19, 29, 33, and 40 after AI were 64%, 54%, 50%, and 45%, respectively. At d 19 after AI, P/AI was less likely in cows diagnosed with ENDO (52% vs. 69%) or PVD (54% vs. 67%). At d 29, P/AI was less likely in cows with Hp ≥1.54 g/L at 2 DIM (38% vs. 55%) or PVD (35% vs. 56%). Both metritis and ENDO were associated with decreased P/AI at d 40 after AI. Cows diagnosed with metritis had greater risk of P-LOSS from d 19 to 29 (43% vs. 22%) or from d 33 to 40 (37% vs. 7%) than cows without metritis. From d 29 to 33, the risk of P-LOSS was greater in cows with NEFA ≥0.73 mM at 2 DIM (13% vs. 5%) or BCS ≤2.75 at 63 DIM (14% vs. 5%). The concentration of P4 on d 8 after AI was positively associated with P/AI at d 29, 33, and 40, and negatively associated with P-LOSS from d 19 to 29. Postpartum health disorders, particularly reproductive tract disease, can have detrimental effects on early pregnancy establishment and on pregnancy maintenance from d 19 to 40 after AI.

Postpartum health is associated with detection of estrus by activity monitors and reproductive performance in dairy cows.

The objective of this prospective observational study was to investigate associations of postpartum health with estrus detection (ED) by activity monitors and pregnancy outcomes in dairy cows. A total of 1,743 Holstein cows from 2 commercial dairy herds in Ontario, Canada were enrolled 3 wk before expected parturition and examined for health variables until 9 wk postpartum. Body condition score (BCS) and lameness were measured at 3 wk prepartum, and serum concentrations of total Ca, haptoglobin (Hp), and nonesterified fatty acids were measured at 2 and 6 ± 2 d in milk (DIM), and blood ß-hydroxybutyrate (BHB) and metritis were assessed at 4, 8, 11, and 15 ± 2 DIM. Cows were examined for purulent vaginal discharge (PVD) and endometritis (ENDO) by endometrial cytology at wk 5, for lameness at wk 3 and 7, for BCS at wk 9 postpartum, and for time to onset of cyclicity by biweekly serum progesterone (P4) measurements. Additional disease data were obtained from farm records. Reproductive management for first AI was primarily based on ED by activity monitors until at least 75 DIM, and cows not detected in estrus were synchronized. Data were analyzed in multivariable logistic or Cox proportional hazards regression models including blood markers, health variables, potential covariates, and herd as a random effect. Estrus was detected in 77% of primiparous and 66% of multiparous cows between 50 or 55 DIM and 75 DIM. In 1,246 cows, the model-predicted probability of ED (percentage point difference) was lower in cows that had retained placenta (-14%), ENDO (-7%), PVD (-8%), delayed cyclicity (no P4 > 1 ng/mL by wk 9; -12%), or ≥0.5-point BCS loss (-14%) compared with cows without each of these risk factors, and it was negatively associated with blood BHB at 15 DIM. Considering only variables measured on farm (not requiring laboratory analysis), the probability of ED was lower (56 vs. 81%) in cows with >1 risk factor compared with cows without risk factors. The predicted probability of pregnancy at first artificial insemination (percentage point difference) was lower in cows that had ENDO (-7%) or PVD (-7%), and negatively associated with serum Hp at 6 ± 2 DIM. In cows detected in estrus by 75 DIM (n = 888), risk factors for reduced pregnancy rate by 250 DIM (adjusted hazard ratio (AHR); 95% confidence intervals) included difficult calving (AHR: 0.67; 0.45 to 1.00), metritis (AHR: 0.79; 0.61 to 1.01), PVD (AHR: 0.79; 0.65 to 0.97), or lameness (AHR: 0.79; 0.62 to 1.01), and it was negatively associated with serum Hp at 6 ± 2 DIM. Monitoring postpartum health may be used to identify cows that are more or less likely to be detected in estrus by activity monitors and to become pregnant in a timely manner. This would support a selective reproductive management program with targeted interventions.

Transcriptome changes associated with elongation of bovine conceptuses I: Differentially expressed transcripts in the conceptus on day 17 after insemination.

The objective was to characterize transcriptome changes associated with elongation in bovine conceptuses during preimplantation stages. Nonlactating Holstein cows were euthanized 17 d after artificial insemination (AI) and the uterine horn ipsilateral to the CL was flushed with saline solution. Recovered conceptuses were classified as small (1.2 to 6.9 cm; n = 9), medium (10.5 to 16.0 cm; n = 9), or large (18.0 to 26.4 cm; n = 10). Total mRNA was extracted and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 634, 240, and 63 differentially expressed transcripts, respectively. Top canonical pathways of known involvement with embryo growth that were upregulated in large conceptuses included actin cytoskeleton (LvsS), integrin signaling (LvsS and LvsM), ephrin receptor (LvsS), mesenchymal transition by growth factor (LvsM), and regulation of calpain protease (LvsS). Transcripts involved with lipid metabolism pathways (LXR/RXR, FXR/RXR, hepatic fibrosis) were associated with the LvsS and LvsM, and some transcripts such as APOC2, APOH, APOM, RARA, RBP4, and PPARGC1A, were involved in these pathways. An overall network summary associated biological downstream effects of invasion of cells, proliferation of embryonic cells, and inhibition of organismal death in the LvsS. In conclusion, differently expressed transcripts in the LvsS comparison were associated with the cell growth, adhesion, and organismal development, although part of these findings could be attributed to differences in circulatory concentrations of progesterone of the cows that bore large and small conceptuses. The large and medium conceptuses developed under similar concentrations of progesterone and presented 240 differently expressed transcripts, associated with cell differentiation, metabolite regulation, and other biological processes.

Transcriptome changes associated with elongation of bovine conceptuses II: Differentially expressed transcripts in the endometrium on day 17 after insemination.

The objective was to characterize endometrial transcriptome on d 17 of gestation in dairy cows according to conceptus length. Nonlactating Holstein cows (n = 48) were slaughtered 17 d after AI and the uterine horn ipsilateral to the corpus luteum (CL) was flushed with saline solution. Recovered conceptuses were classified as small (1.2-6.9 cm; n = 9), medium (10.5-16.0 cm; n = 9), or large (18.0-26.4 cm; n = 10). Samples of intercaruncular endometrium dissected from the caudal, intermediate, and cranial portions of the uterine horn ipsilateral to the pregnancy were pooled for analyses. Total mRNA was extracted from endometrial tissue and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between endometria of cows carrying large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 235, 21, and 94 differentially expressed transcripts, respectively. Top canonical pathways included the antigen presentation pathway and Th1/Th2 activation pathways, both for LvsS and MvsS. Interferon-α and -γ were identified as activated upstream regulators, primarily based on differently expressed transcripts such as IDO1, ISG20, WARS, LGALS9, IFI44, and PSMB9 (LvsS and MvsS). For LvsS, regulator analyses revealed predicted activation of FOXO1, IFN, NFACTC2, IL-12, IL-6, and IL-18, whereas it depicted inhibition of IL10RA and ZBTB1. Changes in these regulators were associated with a downstream activation of leukocytes, as well as quantity and expansion of T lymphocytes. Canonical pathways associated with the comparison LvsM included cell cycle G2/M DNA damage checkpoint regulation, cell cycle control of chromosomal replication. Moreover, tretinoin was predicted, as activated in upstream analysis for the same comparison. In conclusion, most of the differently expressed transcripts in the endometrium on d 17 of gestation were identified between cows carrying small conceptuses compared with counterparts carrying medium and large conceptuses and were involved with pathways associated with modulation of the immune response.

Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows.

Our objectives were to evaluate the effects of complete replacement of inorganic salts of trace minerals (STM) with organic trace minerals (OTM) in both pre- and postpartum diets on ovarian dynamics, estrous behavior measured by sensors, preimplantation conceptus development, and reproductive performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving. Pre- and postpartum diets were formulated to meet 100% of recommended levels of each trace mineral in both treatments, taking into consideration both basal and supplemental levels. The final target concentrations of Co, Cu, Mn, Se, and Zn were, respectively, 0.25, 13.7, 40.0, 0.3, and 40.0 mg/kg in the prepartum diet, and 0.25, 15.7, 40.0, 0.3, and 63.0 mg/kg in the postpartum diet. The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and sodium selenite, while the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 d in milk (DIM) and were assigned to individual cows using automatic feeding gates. Starting at 21 DIM, ultrasonography examinations of the ovaries were performed weekly to determine the presence of a corpus luteum and postpartum resumption of ovarian cyclicity. Cows were presynchronized with 2 injections of PGF2α at 42 and 56 DIM. Estrous behavior was monitored using electronic activity tags that indirectly measured walking activity. Cows detected in estrus after the second PGF2α were inseminated, and those not detected in estrus by 67 DIM were enrolled in a synchronization program. Cows that returned to estrus after artificial insemination (AI) were reinseminated. Pregnancy diagnosis was performed 33 d after AI, and nonpregnant cows were resynchronized. Transcript expression of interferon-stimulated genes in peripheral blood leukocytes was performed in a subgroup of cows (STM, n = 67; OTM, n = 73) on d 19 after AI. A different subgroup of cows (28 STM, 29 OTM) received uterine flushing 15 d after AI for recovery of conceptuses and uterine fluid for analyses of transcriptomics and metabolomics, respectively. In addition, dominant follicle diameter, luteal size and blood flow, and concentration of progesterone in plasma were measured on d 0, 7, and 15 relative to AI. After flushing, PGF2α was given and the dominant follicle was aspirated 2 d later to measure the concentration of trace minerals by mass spectrometry. Estrous behavior, size of the dominant follicle and corpus luteum, concentration of progesterone, time to pregnancy, and proportion of cows pregnant by 100 d of the breeding period did not differ between treatments. A greater proportion of cows supplemented with OTM had a corpus luteum detected before presynchronization (64.3 vs. 75.2%), and primiparous cows supplemented with OTM tended to resume cyclicity earlier than their STM counterparts. Cows supplemented with OTM had a greater concentration of Cu in follicular fluid than cows supplemented with STM (0.89 vs. 0.77 µg/mL, respectively). In pregnant multiparous cows, expression of receptor transporter protein 4 in peripheral blood leukocytes was 42% greater in the OTM group. Conceptuses of the 2 treatments had 589 differentially expressed transcripts, with many indicating advanced conceptus elongation and greater transcript expression of selenoproteins in the OTM group. In pregnant cows, 24 metabolites were more abundant in the uterine fluid of OTM, including spermidine, sucrose, and cholesterol. In conclusion, replacing STM with OTM caused modest improvements to resumption of ovarian cyclicity and important changes in preimplantation conceptus development, but it did not alter conception risk and pregnancy rate.

Effects of replacing inorganic salts of trace minerals with organic trace minerals in the diet of prepartum cows on quality of colostrum and immunity of newborn calves.

Our objectives were to evaluate the impact of supplementary trace mineral (TM) form-inorganic salts (STM; Co, Cu, Mn, Zn sulfates, and Na selenite) or organic (OTM; Co, Cu, Mn, Zn proteinates, and selenized yeast)-in the prepartum diet on quantity and quality of colostrum, passive immunity, antioxidant biomarkers, cytokine responses to lipopolysaccharide (LPS), health, and growth of newborn calves. Pregnant heifers (n = 100) and cows (n = 173) were enrolled at 45 d before calving, blocked by parity and body condition score, and allocated randomly to STM (50 heifers; 86 cows) or OTM (50 heifers; 87 cows) supplementation. Cows in both treatments were fed the same diet, except for the source of supplementary TM. Within 2 h of calving, dams and calves were separated, colostrum was harvested, the yield was measured, and a sample was saved for posterior analyses of colostrum quality. A subgroup of calves (n = 68) had a blood sample collected before colostrum feeding. After colostrum feeding, all samples and data collection were limited to 163 calves (STM = 82; OTM = 81) fed 3 L of good quality (Brix% >22) maternal colostrum via nipple bottle minutes after harvesting. Concentration of IgG in colostrum and serum was determined 24 h after colostrum feeding using radial immunodiffusion. Concentration of TM in colostrum and serum were performed by inductively coupled plasma mass spectrometry. Activity of glutathione peroxidase, ferric reducing ability of plasma, and concentration of superoxide dismutase were evaluated in plasma by colorimetric assays. Ex vivo whole blood stimulation with LPS was performed on d 7 of life to evaluate cytokine responses in a subgroup of 66 calves. Health events were recorded from birth to weaning, and body weight was recorded at birth (all calves) and on d 30 and 60 (heifers only). Continuous variables were analyzed by ANOVA and binary responses were analyzed by logistic regression. Complete replacement of STM by OTM in prepartum diet resulted in greater concentration of Se (461 vs. 543 ± 7 µg/g; ± SEM) but did not alter the concentration or total mass of other TM and IgG in colostrum. Female calves of the OTM group had greater concentration of Se in serum at birth (0.23 vs. 0.37 ± 0.05 µg/mL), were lighter in weight at birth (40.9 vs. 38.8 ± 0.6 kg) and weaning (93.2 vs. 89.7 ± 1.6 kg) than those of the STM group. Maternal treatments did not affect passive immunity or antioxidant biomarkers. On d 7, basal concentrations (log10 of concentration in pg/mL) of IFNγ (0.70 vs. 0.95 ± 0.083) and LPS-stimulated concentrations of CC chemokine ligand 2 (CCL2; 2.45 vs. 2.54 ± 0.026), CC chemokine ligand 3 (CCL3; 2.63 vs. 2.76 ± 0.038), IL-1α (2.32 vs. 2.49 ± 0.054), and IL-1ß (3.62 vs. 3.86 ± 0.067) were greater in OTM than in STM. Supplementation with OTM in pregnant heifers, but not in pregnant cows, reduced the incidence of preweaning health problems in their calves (36.4 vs. 11.5%). Complete replacement of STM by OTM in the prepartum diet did not cause major changes in colostrum quality, passive immunity, and antioxidant capacity, but increased cytokine and chemokine responses to LPS on d 7 of life and benefited preweaning health of calves born to primiparous cows.

Neutrophil function and antibody production during the transition period: Effect of form of supplementary trace minerals and associations with postpartum clinical disease and blood metabolites.

Our objectives were to evaluate the effect of the form of supplementary trace minerals-inorganic salts (STM: Co, Cu, Mn, and Zn sulfates and Na selenite) or organic (OTM: Co, Cu, Mn, Zn proteinates, and selenized yeast)-fed at 100% of recommended levels in both pre- and postpartum diets on in vitro phagocytic activity of neutrophils, and in vivo IgG responses to an ovalbumin challenge during the transition period. In addition, we investigated the associations of these immunological responses with incidence of postpartum clinical diseases and the dynamic changes of metabolic markers during the transition period. Pregnant heifers and cows (n = 273) were enrolled at 45 ± 3 d before expected calving, blocked by parity and body condition score, and allocated randomly to STM or OTM supplementation. Cows in both treatments were fed the same diet, except for the form of supplementary trace minerals. Automatic feeding gates were used to assign treatments to individual cows. Blood was collected on d -7 ± 3 and 7 ± 3 relative to calving in a subgroup of cows (n = 131 and 133, respectively) to measure phagocytic activity of neutrophils in vitro using flow cytometry. Subcutaneous immunization with 0.5 mg of chicken egg ovalbumin was performed in a subgroup of cows (n = 181) on d -45, -21, and 3 relative to calving. Concentration of anti-ovalbumin IgG in serum was measured by ELISA on d -45, -21, 3, 7, and 21. Trace mineral concentrations in blood were measured by inductively coupled plasma mass spectrometry on d -45, -21, -7, 0, 7, and 21 relative to calving. Selected metabolites were measured on d -21, -10, -3, 0, 3, 7, 10, 14, and 21 relative to calving. Treatment did not affect the percentage of neutrophils performing phagocytosis on d -7 or 7 but the median fluorescence intensity of phagocytosis on d 7 was greater for OTM than STM. We found no differences between treatments in the level of anti-ovalbumin IgG in serum on any of the sampling days. Changes in neutrophil function from prepartum to postpartum were associated with incidence of postpartum clinical disease, postpartum feed intake and milk production, concentrations of Ca, K, Se, Mn, Co, and total protein in serum. Immunoglobulin G responses to ovalbumin injections were not associated with incidence of postpartum clinical disease but were associated with body weight, feed intake, energy balance, and concentrations of nonesterified fatty acids, aspartate aminotransferase, gamma-glutamyl transpeptidase, albumin, Na, P, and Cu in serum. In conclusion, replacement of STM by OTM improved one measure of phagocytic capacity of neutrophils in vitro, which was also greater in cows that did not develop postpartum clinical disease. The associations of innate and acquired immune responses with feed intake, energy balance, and circulating concentrations of key macro and micronutrients reinforce the importance of nutritional management for the health of dairy cows during the transition period.

Effects of replacing inorganic salts of trace minerals with organic trace minerals in pre- and postpartum diets on feeding behavior, rumen fermentation, and performance of dairy cows.

Our objectives were to evaluate the effects of complete replacement of supplementary inorganic salts of trace minerals (STM) by organic trace minerals (OTM) in both pre- and postpartum diets on feeding behavior, ruminal fermentation, rumination activity, energy metabolism, and lactation performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving date. Both groups received the same diet, except for the source of trace minerals (TM). The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and Na selenite, whereas the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 days in milk and pre- and postpartum diets were formulated to meet 100% of recommended levels of each TM in both treatments, taking into consideration both basal and supplemental levels. Automatic feed bins were used to assign treatments to individual cows and to measure feed intake and feeding behavior. Rumination activity was monitored by sensors attached to a collar from wk -3 to 3 relative to calving. Blood metabolites were evaluated on d -21, -10, -3, 0, 3, 7, 10, 14, 23, and 65 relative to calving. Ruminal fluid samples were collected using an ororuminal sampling device on d -21, 23, and 65 relative to calving, for measurement of ruminal pH and concentration of volatile fatty acids. Cows were milked twice a day and milk components were measured monthly. Cows supplemented with OTM tended to have longer daily feeding time (188 vs. 197 min/d), and greater dry matter intake (DMI; 12.9 vs. 13.3 kg), and had a more positive energy balance (3.6 vs. 4.2 Mcal/d) and shorter rumination time per kg of dry matter (DM; 40.1 vs. 37.5 min/kg of DM) than cows supplemented with STM during the prepartum period. In the postpartum period, OTM increased DMI in multiparous cows (24.1 vs. 24.7 kg/d) but not in primiparous cows (19.1 vs. 18.7 kg/d). The difference in DMI of multiparous cows was more evident in the first 5 wk of lactation, when it averaged 1 kg/d. Milk yield was not affected by treatment in multiparous cows (44.1 vs. 44.2 kg/d); however, primiparous cows supplemented with OTM had lesser yields than primiparous cows supplemented with STM (31.9 vs. 29.8 kg/d). Cows supplemented with OTM had a greater percentage of protein in milk (3.11 vs. 3.17%), reduced concentration of nonesterified fatty acids in serum (0.45 vs. 0.40 mmol/L), and rumination activity (30.1 vs. 27.8 min/kg of DM) than cows supplemented with STM. At the end of the transition period, cows supplemented with OTM had reduced molar proportion of acetate, reduced pH, and tended to have a greater concentration of total volatile fatty acids in ruminal fluid. In conclusion, complete replacement of STM by OTM caused modest changes in rumen fermentation, feeding behavior, energy metabolism, and performance of dairy cows, improving postpartum DMI in multiparous cows and reducing circulating levels of nonesterified fatty acids. The pre-absorptive effects of TM source and the parity specific responses on performance warrant further research.

Effects of progesterone concentrations and follicular wave during growth of the ovulatory follicle on conceptus and endometrial transcriptome in dairy cows.

Objectives were to evaluate the effects of follicular wave and progesterone concentration on growth of the ovulatory follicle, conceptus elongation, uterine IFN-τ concentration, and transcriptome of conceptus and endometrium of pregnant cows on d 17 of gestation. Nonlactating nonpregnant Holstein cows were assigned randomly to one of 3 treatments: ovulation of a first-wave follicle (FW, n = 15); ovulation of a first-wave follicle and progesterone supplementation (FWP4, n = 12); and ovulation of a second-wave follicle (SW, n = 19). Ovulation of a first- or second-wave follicle was achieved by initiating the Ovsynch protocol (d -9 GnRH, d -2 and -1 PGF2α, d 0 GnRH and artificial insemination, d 0.7 artificial insemination) on d 0 or 6 of a presynchronized estrous cycle, respectively. Cows in FWP4 received 3 intravaginal inserts containing progesterone at 12, 24, and 48 h after the first GnRH injection that were removed on d -2. Cows were killed on d 17 for collection of the reproductive tract. Transcriptome was evaluated by microarray using the Affymetrix Bovine Array. Orthogonal contrasts were built to assess the effects of progesterone concentration during follicle growth (FW vs. FWP4 + SW) and follicular wave (FWP4 vs. SW). Progesterone concentrations (LSM ± SEM) from d -9 to -2 were greater for SW, followed by FWP4 and FW (5.38 ± 0.24, 4.26 ± 0.28, and 1.17 ± 0.27 ng/mL). Diameter of the ovulatory follicle (FW = 19.6 ± 0.6; FWP4 = 15.6 ± 0.6; SW = 15.2 ± 0.5 mm) and concentrations of estradiol from d -2 to 1 (FW = 4.05 ± 0.33; FWP4 = 2.73 ± 0.35; SW = 2.48 ± 0.30 pg/mL) were greater for FW compared with FWP4 and SW. Progesterone concentrations from d 3 to 16 were greater for FW compared with FWP4 and SW. A total of 28 singleton conceptuses were collected (FW, n = 8; FWP4, n = 8; SW, n = 12) and only intact conceptuses were included in the analyses of length (FW, n = 8; FWP4, n = 6; SW, n = 12). Although conceptuses were longer for FW compared with FWP4 and SW (FW = 16.6 ± 2.3; FWP4 = 9.8 ± 2.2; SW = 9.6 ± 2.0 cm), treatment did not affect the amount of IFN-τ in uterine flushing. Transcriptome of conceptuses and endometrium of pregnant cows was not extensively affected by follicular wave (8 and 1 differentially expressed transcripts) or concentration of progesterone during follicle growth (0 and 3 differentially expressed transcripts), showing that these factors did not affect conceptuses and endometrium transcriptome in pregnancies that are maintained to d 17.

Dynamics of lipid droplets in the endometrium and fatty acids and oxylipins in the uterine lumen, blood, and milk of lactating cows during diestrus.

Our objective was to investigate the lipid content of uterus, blood plasma, and milk at early, mid, and late diestrus. Lactating cows (n = 30) had the estrous cycle and ovulation synchronized by administration of exogenous hormones. Cows were blocked by parity and assigned randomly to receive transcervical uterine flushing and biopsy on d 5 (early diestrus), 10 (mid diestrus) or 15 (late diestrus) of the estrous cycle. Flushing and endometrial biopsy were performed in the uterine horn ipsilateral to the corpus luteum. The recovered flushing was used for analyses of lipid composition by liquid chromatography-tandem mass spectrometry and the biopsy was used for investigation of lipid droplet abundance in endometrial cryosections using a neutral lipid fluorescent dye. In addition, blood and milk samples were collected from all cows on d 5, 10, and 15. All blood samples were used to measure the concentration of progesterone in plasma, and all milk samples were used to determine milk composition. Subsamples of blood plasma and milk were also used to evaluate the composition of fatty acids and oxylipins using the same methodology used for uterine flushing samples. The abundance of lipid droplets in the endometrium increased 1.9-fold from d 5 to 10, and 2-fold from d 10 to 15. Concentration of long-chain fatty acids and oxylipins in uterine flushing were, on average, 2.2 and 2.5 times greater in samples collected on d 15 compared with those collected on d 5 and 10. These differences were not observed in blood and milk, suggesting that accumulation of fatty acids and oxylipins in the uterus is regulated locally. In addition to concentration, the profile of individual fatty acids and oxylipins in uterine lumen changed substantially during diestrus. The main categories with increased abundance at late diestrus were mono- and polyunsaturated fatty acids, and oxylipins derived from arachidonic acid, dihomo-γ-linolenic acid, and docosahexaenoic acid. In conclusion, fatty acids and oxylipins accumulate in the uterine lumen during diestrus and might work as a mechanism to supply these lipids to the developing conceptus at late diestrus, when the onset of elongation occurs and substantial synthesis of biomass and cell signaling by lipid mediators are required.

Effect of anti-inflammatory treatment on systemic inflammation, immune function, and endometrial health in postpartum dairy cows.

Systemic inflammation (SI) is increasingly studied in several species because it may be central in many metabolic disturbances and be a risk factor for clinical disease. This proof-of-concept study evaluated the effects of the anti-inflammatory drug meloxicam on markers of SI and energy metabolism, polymorphonuclear neutrophil (PMN) function, and endometritis in clinically healthy postpartum dairy cows. Cows received meloxicam (0.5 mg/kg of body weight; n = 20) once daily for 4 days (10-13 days postpartum) or were untreated (n = 22). Blood samples were collected -7, 1, 3, 5, 7, 10, 11, 12, 13, 14, 18, 21, 28, and 35 days relative to calving to measure serum concentrations of metabolic and inflammatory markers. Function of peripheral blood PMN were evaluated at 5, 10, 14, and 21, and proportion of PMN in endometrial cytology were performed at 5, 10, 14, 21, 28 and 35 days postpartum. Meloxicam decreased serum haptoglobin from the second until the last day of treatment, and improved indicators of energy metabolism (lesser ß-hydroxybutyrate and greater insulin-like growth factor-1 during treatment, and greater glucose at the end of treatment than control cows). This improved PMN function at 14 days postpartum, but the endometrial inflammatory status was not affected.

Associations between maternal characteristics and health, survival, and performance of dairy heifers from birth through first lactation.

The objective of this study was to investigate whether health, survival, and performance of dairy heifers from birth through first lactation are associated with parity and health status of their dams. Holstein heifers (n = 1,811) derived from artificial insemination were categorized as (1) daughters of primiparous cows that, consequently, were nonlactating heifers during gestation (Prim-NoL; n = 787); (2) daughters of multiparous cows that did not have any clinical diseases in the previous lactation (Mult-NoCD; n = 638); and (3) daughters of multiparous cows that had at least one clinical disease in the previous lactation (Mult-CD; n = 386). Clinical diseases of the multiparous dams included retained placenta, metritis, mastitis, lameness, and digestive and respiratory problems. Data collected for evaluation of daughters included genotypic and phenotypic characteristics at birth, morbidity, reproductive performance, and culling from birth through 305 d in milk of first lactation. Orthogonal contrasts were used to evaluate the effect of the parity of the dam (Prim-NoL vs. Mult-NoCD + Mult-CD) and the effect of clinical disease occurrence in the previous lactation among multiparous dams (Mult-NoCD vs. Mult-CD). Compared with daughters of multiparous cows, daughters of Prim-NoL were lighter at birth (36 vs. 41 kg), had greater genetic merit for production traits (e.g., genomic estimated breeding value for milk yield: 875 vs. 746 kg), were less likely to leave the herd (17 vs. 28%) and to lose pregnancy as a heifer (9 vs. 14%), calved earlier (703 vs. 711 d old), were less likely to have clinical diseases as a first lactation cow (30 vs. 37%), and had reduced performance in the first lactation when considering their genetic merit (e.g., 305-d yield of energy-corrected milk: 11,270 vs. 11,539 kg). Compared with daughters of Mult-NoCD, daughters of Mult-CD were less likely to have digestive problems as a heifer (17 vs. 27%) and clinical disease as a first lactation cow (32 vs. 42%), but were also more likely to leave the herd as a heifer (32 vs. 25%) even though genetic merit for production traits were similar (e.g., genomic estimated breeding value for milk: 744 vs. 749 kg). In conclusion, both parity and health status of the dam in the previous lactation were associated with morbidity, survival, and performance of their daughters from birth through 305 d in milk of the first lactation and might represent factors affecting developmental programming of dairy heifers in utero.

Long-term effects of postpartum clinical disease on milk production, reproduction, and culling of dairy cows.

Two retrospective studies examining data of 7,500 lactating cows from a single herd were performed with the objective of evaluating the long-term effects of clinical disease during the early postpartum period on milk production, reproduction, and culling of dairy cows through 305 days in milk (DIM). In the first study, data regarding health, milk production, reproduction, and culling of 5,085 cows were summarized. Cows were classified according to incidence of clinical problem (metritis, mastitis, lameness, digestive problem, or respiratory problem) during the first 21 DIM (ClinD21). During 305 d of lactation, cows that had ClinD21 produced, on average, 410 kg less milk, 17 kg less fat, and 12 kg less protein compared with cows that did not have ClinD21 (NoClinD21). Although the interval to first breeding was not different between groups of interest, pregnancy rate through 305 DIM was lower in cows that had ClinD21 [adjusted hazard ratio (AHR) = 0.81]. When individual breedings were analyzed, cows that had ClinD21 presented lower rates of pregnancy per breeding for breedings performed before 150 DIM, reduced numbers of calving per breeding for breedings performed before 200 DIM, and greater number of pregnancy losses for all breedings performed through 305 DIM. The rate of culling from calving through 305 DIM was higher in cows that had a single ClinD21 (AHR = 1.79) and in cows that had multiple ClinD21 (AHR = 3.06), which resulted in a greater proportion of cows leaving the herd by 305 DIM (NoClinD21 = 22.6%; single ClinD21 = 35.7%; multiple ClinD21 = 53.8%). In the second study, data regarding postpartum health and 305-d yields of milk, fat, and protein were collected from 2,415 primiparous cows that had genomic testing information. Genomic estimated breeding values (EBV) were used to predict 305-d yields of milk, fat, and protein. Genomic EBV and predicted yields of milk, fat, and protein did not differ between cows that had ClinD21 and those that did not have ClinD21. In contrast, the observed 305-d yields of milk, fat, and protein were reduced by 345, 10, and 10 kg, respectively, in cows that had ClinD21 compared with cows that did not have ClinD21. We conclude that clinical disease diagnosed and treated during the first 21 DIM has long-term effects on lactation performance, reproduction, and culling of dairy cows, which contribute to detrimental consequences of health problems on sustainability of dairy herds. Replication of our studies in multiple herds will be important to confirm our findings in a larger population.

Feed restriction to induce and meloxicam to mitigate potential systemic inflammation in dairy cows before calving.

Most dairy cows experience a transient decrease in feed intake in the 1 to 2 wk before calving, which has been associated with systemic inflammation (SI), indicated by increased blood haptoglobin (Hp) concentration. We aimed to characterize the association between prepartum decrease in feed intake and the onset of SI and, if present, the ability of meloxicam (MEL), a non-steroidal anti-inflammatory drug, to mitigate SI. Holstein cows (n = 45) were assigned to control (n = 13), feed restriction (FR) untreated (FR-U; n = 15), and FR treated with MEL (FR-T; n = 17) groups. Daily feed intake was measured from -22 d from expected parturition until 35 d postpartum. Control cows were fed ad libitum, whereas FR-U and FR-T cows were reduced to 60% of their average intake for 4 consecutive days (-15 to -12 d from expected calving). The FR-T cows received MEL (0.5 mg/kg of body weight) once daily for 4 consecutive days (-13 to -10 d from expected calving). Blood samples were collected -22, -15, -14, -13, -12, -10, -7, -5, -3, 0, 1, 3, 5, 7, 15, 22, and 35 d relative to calving to measure serum concentrations of total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, ß-hydroxybutyrate, nonesterified fatty acids, Hp, and insulin-like growth factor-1. Serum concentrations of lipopolysaccharide-binding protein were measured -22, -15, -14, -13, -12, and -10 d from expected calving. Simplified glucose tolerance tests were performed on -15, -12, -5, 1, and 5 d relative to calving. Mixed linear regression models were used to assess the effects of FR and MEL on each metabolite. The interaction between treatment group and blood sampling day was forced into each model. All models accounted for body condition score, parity, and the cow as a random effect. Nonesterified fatty acids concentrations in both the FR-U and FR-T groups significantly increased from the second until the last day of FR. Feed restriction increased urea concentrations compared with the control group on -14 d but decreased urea concentrations on -10 d from expected calving. Control cows had greater ß-hydroxybutyrate concentrations compared with FR cows on 15, 21, and 35 d postpartum. For all other metabolites, no differences were found. This model of FR produced substantial fat mobilization but based on serum Hp and lipopolysaccharide-binding protein concentrations did not generate measurable SI; therefore, we were unable to evaluate the ability of MEL to mitigate SI.

Effect of dietary transition at dry off on the behavior and physiology of dairy cows.

This study (1) investigated the effect of dietary transition at dry off on the behavior and physiology of Holstein dairy cows and (2) evaluated the efficacy of utilizing reductions in dietary nutrient density and milking frequency to mediate the dry-off process. Forty-eight cows were dried off over a 5-d period approximately 60 d before their expected calving dates. At the start of dry off (d 1), cows were randomly assigned to 1 of 2 total mixed rations that differed in nutrient density: higher nutrient density (HND; n = 24) and lower nutrient density (LND; n = 24). During dry off, cows were milked 1×/d on d 1, 2, and 3 and a final time on d 5. Milk yield, feeding behavior, rumination, lying behavior, and reticulorumen pH were monitored electronically and feed samples were collected to determine feed sorting. Blood samples, body condition scores, and body weights were taken periodically. Across treatments, milk yield was reduced by approximately 10 kg/d during dry off, with cows fed the LND diet producing 0.9 kg less milk at the time of dry off compared with cows fed the HND diet. Cows fed the LND diet consumed 2.2 kg less dry matter per day, fed at a slower rate, spent 15.7 min more feeding, and sorted to a greater extent for the smaller, more nutrient dense components of the diet compared with cows fed the HND diet. Despite sorting activity, cows fed the LND diet ruminated 3.3 min more per kilogram of dry matter consumed and had a mean reticulorumen pH 0.07 units higher compared with cows fed the HND diet. Following the start of dry off, cows spent approximately 5% more of their free time lying down. Dietary transition and dry off similarly affected the energy balance and inflammatory response of cows, although these parameters varied by day after the start of dry off. The results of this study indicate that the LND diet more effectively reduced milk production before dry off compared with the HND diet while not substantially altering cow physiology.

Using in-line milk progesterone data to characterize parameters of luteal activity and their association with fertility in Holstein cows.

Our objectives were to characterize parameters of luteal activity based on milk progesterone concentration (P4c) data from before and after artificial insemination (AI) and to evaluate their potential association with fertility in Holstein cows. Records of AI events (n = 4,353) and of milk P4c (n = 158,961) obtained through an in-line milk analysis system (Herd Navigator, DeLaval International, Tumba, Sweden) from 1,891 lactations of 1,423 Holstein cows were evaluated. Milk P4c (ng/mL) were measured every 2.2 ± 1.9 d (mean ± standard deviation) between 23.6 ± 7.3 and 185.3 ± 56.7 d in milk. Variations in milk P4c of consecutive records were used to determine onset of luteal phase (increase in P4c from <5.0 to ≥5.0 ng/mL), luteal phase length (period, in days, of P4c ≥5.0 ng/mL), cessation of luteal phase (decline from ≥5.0 to <5.0 ng/mL, designated as P4c-decline), and pregnancy (AI followed by a luteal phase that remained uninterrupted until 50 d post-AI). The length of the luteal phase preceding AI, the highest P4c (P4c peak) during the luteal phase preceding AI, the lowest P4c preceding AI (P4c pre-AI) that followed a P4c-decline, and the interval between P4c-decline and AI were evaluated, as well as the interval between AI and onset of luteal phase, and P4c at early diestrus (4.5 ± 0.6 d post-AI), mid diestrus (10.0 ± 0.6 d post-AI), and late diestrus (14.1 ± 0.6 d post-AI). Data were analyzed using logistic regressions, and comparisons made based on quartiles and cut-points established by receiver operating characteristic curve analysis. Overall probability of pregnancy was 32.0%. Parameters associated with reduced probability of pregnancy (represented as percentage points decrease in the probability of pregnancy) were (1) luteal phase length >14.4 d (7.6% decrease), (2) P4c peak ≤24.7 ng/mL (4.5% decrease), (3) P4c pre-AI >0.5 ng/mL (5.5% decrease), (4) interval between P4c-decline and AI of >1.6 d (4.0% decrease), (5) interval between AI and onset of luteal phase of <7 or >11 d (9.3 and 12.1% decrease, respectively), and (6) P4c at early diestrus ≤0.7 or >3.5 ng/mL (15.2 and 6.7% decrease, respectively), (7) P4c at mid diestrus ≤12.4 ng/mL (12.5% decrease), and (8) P4c at late diestrus ≤22.7 ng/mL (9.7% decrease). The parameters of luteal activity associated with reduced probability of pregnancy established here could be used as benchmarks while developing recommendations to improve reproductive performance in herds using in-line milk progesterone monitoring.

Pyrethroid and organophosphate pesticide resistance in field populations of horn fly in Brazil.

Pesticides are used worldwide to control arthropod parasites in cattle herds. The indiscriminate and/or inappropriate use of pesticides without veterinary guidance is a reality in several countries of South America. Improper pesticide use increases the chances of contamination of food and the environment with chemical pesticides and their metabolites. Reduction of these contamination events is an increasing challenge for those involved in livestock production. The horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), is one of the most economically important parasites affecting cattle herds around the world. As such, horn fly control efforts are often required to promote the best productive performance of herds. Pesticide susceptibility bioassays revealed that pyrethroid resistance was widespread and reached high levels in horn fly populations in the Brazilian state of Rondônia. The knockdown resistance (kdr) sodium channel gene mutation was detected in all horn fly populations studied (n = 48), and the super kdr sodium channel gene mutation was found in all homozygous resistant kdr individuals (n = 204). Organophosphate resistance was not identified in any of the fly populations evaluated.