A. Vieira-Neto

Carryover effect of postpartum inflammatory diseases on developmental biology and fertility in lactating dairy cows

The objective of this series of studies was to investigate the effects of inflammatory diseases occurring before breeding on the developmental biology and reproductive responses in dairy cows. Data from 5 studies were used to investigate different questions associating health status before breeding and reproductive responses. Health information for all studies was composed of the incidence of retained fetal membranes, metritis, mastitis, lameness, and respiratory and digestive problems from parturition until the day of breeding. Retained placenta and metritis were grouped as uterine disease (UTD). Mastitis, lameness, digestive and respiratory problems were grouped as nonuterine diseases (NUTD). Study 1 evaluated the effect of disease before artificial insemination (AI), anovulation before synchronization of the estrous cycle, and low body condition score at AI on pregnancy per AI, as well as their potential interactions or additive effects. Study 2 investigated the effect of site of inflammation (UTD vs. NUTD) and time of occurrence relative to preantral or antral stages of ovulatory follicle development, and the effect of UTD and NUTD on fertility responses of cows bred by AI or by embryo transfer. Study 3 evaluated the effect of disease on fertilization and embryonic development to the morula stage. Study 4 evaluated the effect of disease on preimplantation conceptus development as well as secretion of IFN-τ and transcriptome. Study 5 investigated the effect of diseases before AI on the transcript expression of interferon-stimulated genes in peripheral blood leukocytes during peri-implantation stages of conceptus development after first AI postpartum. Altogether, these studies demonstrated that inflammatory disease before breeding reduced fertilization of oocytes and development to morula, and impaired early conceptus development to elongation stages and secretion of IFN-τ in the uterine lumen. Diseases caused inflammation-like changes in transcriptome of conceptus cells, increased risk of pregnancy loss, and reduced pregnancy or calving per breeding. Moreover, the effects on reproduction were independent of cyclic status before synchronization of the estrous cycle and body condition score at breeding, which all had additive negative effects on fertility of dairy cows. Occurrence of disease at preantral or at antral stages of ovulatory follicle development had similar detrimental effects on pregnancy results. The carryover effects of diseases on developmental biology might last longer than 4 mo. Reduced oocyte competence is a likely reason for carryover effects of diseases on developmental biology, but impaired uterine environment was also shown to be involved.

Efficacy of ampicillin trihydrate or ceftiofur hydrochloride for treatment of metritis and subsequent fertility in dairy cows

Our objectives were to evaluate the efficacy of ampicillin trihydrate for the treatment of metritis in dairy cows compared with ceftiofur hydrochloride and the subsequent effects on pregnancy at first insemination (P/AI). Cows in the first 12 d in milk (DIM) with a uterine discharge score of 5 (watery, reddish or brownish discharge of foul smell) and rectal temperature <39.5°C were diagnosed with metritis based on the fetid discharge, and cows with metritis and rectal temperature ≥39.5°C were diagnosed as having puerperal metritis. Cows with metritis (n=528) were blocked by parity and type of metritis as fetid discharge or puerperal metritis and, within each block, assigned randomly to receive 11mg/kg of ampicillin (n=259) or 2.2mg/kg of ceftiofur (n=269) once daily for 5 d. Day of diagnosis of metritis was considered study d 1. A cohort of 268 cows without metritis was selected randomly at 12 DIM. Rectal temperature was measured in cows with metritis on study d 1 to 7, and 12, and vaginal discharge was scored on study d 5, 7, and 12. Metritis cure was characterized by vaginal discharge score of <5 or by vaginal discharge score of <5 and no fever. At 32±3 DIM, vaginal discharge was scored for diagnosis of purulent vaginal discharge. At 39±3 DIM, endometrial cytology was performed. At 53±3 and 67±3 DIM, ovaries were scanned to determine estrous cyclicity. Pregnancy was evaluated after the first AI. Cure of metritis based on vaginal discharge <5 was greater for ampicillin than ceftiofur on d 5 (37.1 vs. 25.2%) and 7 (57.2 vs. 46.3%), but not on d 12 (82.0 vs. 85.0%). Cure of metritis based on vaginal discharge <5 and no fever was greater for ampicillin than for ceftiofur only on d 7 (50.4 vs. 37.9%), but not on d 5 (23.1 vs. 17.6%) and 12 (66.1 vs. 67.4%). Cows with puerperal metritis had reduced cure compared with cows with fetid discharge on d 5 (30.5 vs. 12.8%), 7 (55.2 vs. 33.6%), and 12 (72.0 vs. 61.1%). The proportion of cows with fever on any day after therapy started did not differ between treatments. Fifty-three percent of cows with metritis based on fetid discharge developed fever after initiating antimicrobial therapy. Cows receiving ampicillin had less prevalence of purulent vaginal discharge than those treated with ceftiofur (57.7 vs. 67.8%), but they were both greater than cows without metritis (21.9%). Prevalence of cytological endometritis did not differ between ampicillin and ceftiofur (30.0 vs. 25.4%), but they were both greater than cows without metritis (14.5%). The proportion of estrous cyclic cows (75.0%) and P/AI did not differ among treatments (ampicillin=28.0% vs. ceftiofur=28.3% vs. without metritis=30.5%). Clinical cure was faster for ampicillin than for ceftiofur, but on study d 12 both treatments resulted in similar cure. Clinical cure was less for cows with puerperal metritis than for cows with fetid uterine discharge. Despite differences in uterine health, P/AI at the first insemination did not differ among treatments.

Uterine Microbiota Progression from Calving until Establishment of Metritis in Dairy Cows

ABSTRACT The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as “Candidatus Blochmannia,” Escherichia, Sneathia, and Pedobacter.

Factors associated with early cyclicity in postpartum dairy cows.

The objective of this study was to evaluate factors associated with resumption of ovarian cyclicity within 21 days in milk (DIM) in dairy cows. Cows (n=768) from 2 herds in north Florida had their ovaries scanned at 17±3, 21±3, and 24±3 DIM. Cows that had a corpus luteum ≥20mm at 17±3 or at 21±3 DIM or that had a corpus luteum <20mm in 2 consecutive examinations were determined to be cyclic by 21±3 DIM. The following information was collected for up to 14 DIM: calving season, parity, calving problems, metabolic problems, metritis, mastitis, digestive problems, lameness, body weight loss, dry period length, and average daily milk yield. Body condition was scored at 17±3 DIM. Multivariable mixed logistic regression analysis was performed using the GLIMMIX procedure of SAS. Variables with P≤0.2 were considered in each model. Herd was included as a random variable. Three models were constructed: model 1 included all cows, model 2 included only cows from dairy 1 that had daily body weights available, and model 3 included only multiparous cows with a previous dry period length recorded. In model 1, variables associated with greater cyclicity by 21±3 DIM were calving in the summer and fall rather than in the winter or spring, being multiparous rather than primiparous, and not having metabolic or digestive problems. In model 2, variables associated with greater cyclicity by 21±3 DIM were calving in the summer and fall, not having metritis or digestive problems and not losing >28 kg of BW within 14 DIM. In model 3, variables associated with greater cyclicity by 21±3 DIM were absence of metabolic problems and dry period ≤76 d. In summary, cyclicity by 21±3 DIM was negatively associated with calving in winter or spring, primiparity, metritis, metabolic or digestive problems, loss of >28 kg of body weight, and a dry period >76d. Strategies preventing extended dry period length and loss of BW, together with reductions in the incidence of metritis as well as metabolic and digestive problems should improve early cyclicity postpartum.

Use of calcitriol to maintain postpartum blood calcium and improve immune function in dairy cows

Our objectives were to determine the effects of an injectable formulation of calcitriol on mineral metabolism and immune function in postpartum Holstein cows that received an acidogenic diet prepartum to minimize hypocalcemia. In experiment 1, cows within 6 h of calving received calcitriol (0, 200, or 300 μg) to determine the dose needed to increase plasma concentrations of Ca; 300 μg was sufficient to sustain Ca for at least 3 d. In experiment 2, multiparous cows were assigned randomly to receive only vehicle (control, n = 25) or 300 μg of calcitriol (n = 25) subcutaneously within the first 6 h after calving. Blood was sampled before treatment and 12 h later, then daily until 15 d in milk (DIM), and analyzed for concentrations of ionized Ca (iCa), total Ca (tCa), total Mg (tMg), and total P (tP), metabolites, and hormones. Urine was sampled in the first 7 DIM and analyzed for concentrations of tCa, tMg, and creatinine. Neutrophil function was evaluated in the first week postpartum. Dry matter intake and production performance were evaluated for the first 36 DIM. Calcitriol administration increased concentrations of calcitriol in plasma within 12 h of application from 51 to 427 pg/mL, which returned to baseline within 5 d. Concentrations of iCa and tCa increased 24 h after treatment with calcitriol. Concentrations of iCa (control = 1.08 vs. calcitriol = 1.20 mM), tCa (control = 2.23 vs. calcitriol = 2.33 mM), and tP (control = 1.47 vs. calcitriol = 1.81 mM) remained elevated in cows treated with calcitriol until 3, 5, and 7 DIM, respectively, whereas concentration of tMg (control = 0.76 vs. calcitriol = 0.67 mM) was less in calcitriol cows than control cows until 3 DIM. Concentrations of parathyroid hormone decreased in calcitriol cows compared with control cows (control = 441 vs. calcitriol = 336 pg/mL). Calcitriol tended to increase plasma concentrations of β-hydroxybutyrate and serotonin, but concentrations of glucose, nonesterified fatty acids, and C-telopeptide of type I collagen in plasma did not differ between treatments. Cows treated with calcitriol excreted more urinary tCa (control = 0.5 vs. calcitriol = 2.1 g/d) and tMg (control = 4.5 vs. calcitriol = 5.0 g/d) in the first 7 and 2 DIM, respectively, than control cows. Compared with control, calcitriol improved the proportion of neutrophils with oxidative burst (control = 31.9 vs. calcitriol = 40.6%), mean fluorescence intensity for oxidative burst (control = 90,900 vs. calcitriol = 99,746), and mean fluorescence intensity for phagocytosis (control = 23,887 vs. calcitriol = 28,080). Dry matter intake, yields of milk, and milk components did not differ between treatments. Administration of 300 μg of calcitriol at calving was safe and effective in increasing blood concentration of iCa and plasma concentrations of calcitriol, tCa, and tP for the first 6 d after treatment, and improved measures of innate immune function in early-lactation Holstein cows.

Uterine Microbiota and Immune Parameters Associated with Fever in Dairy Cows with Metritis

Objective This study aimed to evaluate bacterial and host factors causing a fever in cows with metritis. For that, we investigated uterine microbiota using a metagenomic sequencing of the 16S rRNA gene (Study 1), and immune response parameters (Study 2) in metritic cows with and without a fever. Principal Findings (Study1) Bacterial communities were similar between the MNoFever and MFever groups based on distance metrics of relative abundance of bacteria. Metritic cows showed a greater prevalence of Bacteroidetes, and Bacteroides and Porphyromonas were the largest contributors to that difference. A comparison of relative abundance at the species level pointed to Bacteroides pyogenes as a fever-related species which was significantly abundant in the MFever than the MNoFever and Healthy groups; however, absolute abundance of Bacteroides pyogenes determined by droplet digital PCR (ddPCR) was similar between MFever and MNoFever groups, but higher than the Healthy group. The same trend was observed in the total number of bacteria. Principal Findings (Study2) The activity of polymorphonuclear leukocyte (PMN) and the production of TNFα, PGE2 metabolite, and PGE2 were evaluated in serum, before disease onset, at 0 and 3 DPP. Cows in the MNoFever had decreased proportion of PMN undergoing phagocytosis and oxidative burst compared with the MFever. The low PMN activity in the MNoFever was coupled with the low production of TNFα, but similar PGE2 metabolite and circulating PGE2. Conclusion/Significance Our study is the first to show a similar microbiome between metritic cows with and without a fever, which indicates that the host response may be more important for fever development than the microbiome. Bacteroides pyogenes was identified as an important pathogen for the development of metritis but not fever. The decreased inflammatory response may explain the lack of a febrile response in the MNoFever group.

Association among gestation length and health, production, and reproduction in Holstein cows and implications for their offspring

Objectives were to evaluate associations among gestation length (GL) and performance in Holstein cows and their offspring. A total of 8,095 Holstein cows and 3,635 female offspring born alive on 2 farms using only artificial insemination (AI) were evaluated. Gestation length averaged 276 ± 6 d in the 8,095 dams, and it was categorized as short (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), or long (LGL; at least 1 SD above the population mean; mean = 285 d, range 283 to 296 d). Responses evaluated in dams included incidence of diseases within 90 d in milk (DIM), pregnancy at first AI and by 300 DIM, and time to pregnancy. Milk yield and removal from the herd by culling or death were recorded for the first 300 DIM. Responses evaluated in female offspring born alive included removal from the herd and reproductive performance. Within primiparous cows, those with SGL had greater incidence of stillbirth, retained placenta, and metritis than primiparous with AGL or LGL; however, within multiparous cows, those with SGL or LGL had greater incidence of dystocia, stillbirth, retained placenta, and metritis than cows with AGL. Morbidity and rate of morbidity were greater for SGL and LGL than AGL. Rate of removal of dams from the herd was 38% faster for SGL than AGL. Milk production was greatest in AGL cows, but responses depended on parity. For primiparous cows, milk production was less in SGL and LGL than in AGL (AGL = 35.4, SGL = 34.6, LGL = 33.0 ± 0.4 kg/d), whereas for multiparous cows, production was less in SGL but greater in LGL than in AGL (AGL = 41.6, SGL = 38.6, LGL = 42.4 ± 0.3 kg/d). A smaller proportion of cows with SGL received at least one AI, but pregnancy at first AI did not differ among groups. Rate of pregnancy was 11% slower for LGL multiparous than for AGL multiparous. By 300 DIM, pregnancy was greater in AGL than SGL. Pregnancy by 300 DIM in multiparous cows was also greater for AGL than LGL. Heifers from dams with GL that deviated from AGL had greater mortality postweaning (AGL = 3.2 vs. SGL = 6.5 vs. LGL = 5.4%). The rate of removal from the herd was greater for SGL (adjusted hazard ratio = 1.78; 95% CI: 1.26 to 2.52) and LGL (adjusted hazard ratio = 2.00; 95% CI: 1.45 to 2.76) than for AGL heifers. Pregnancy at first AI was lowest for LGL and by 500 d of age a larger proportion of AGL heifers were pregnant than LGL (AGL = 82.3 vs. SGL = 79.2 vs. LGL = 74.0%). Cows with GL within 1 SD of the population mean (270 to 282 d) had improved health, production, and reproduction. Heifers from cows with GL within 1 SD of the population mean had improved health and reproduction. Gestation length affects performance of both dams and their offspring.

Vulvovaginal laceration as a risk factor for uterine disease in postpartum dairy cows

The main objective was to evaluate the association between vulvovaginal laceration and uterine diseases in dairy cows. The secondary objectives were to evaluate the association between vulvovaginal laceration and cyclicity, and reproductive performance. The vulvovaginal region of 660 Holstein cows from a 5,000 lactating-cows herd was inspected at 4d in milk (DIM) for the presence of lacerations, and scored (VLS) as follows: 0=no laceration; 1=laceration <2cm at the dorsal commissure of the vulva or lateral walls of the vulva/vagina; 2=laceration ≥2cm at the dorsal commissure of the vulva or at the lateral walls of the vulva/vagina, or both. Vaginal discharge was scored at 4, 6, and 8 DIM for diagnosis of metritis, and then at 32 DIM for diagnosis of purulent vaginal discharge (PVD). Data were analyzed using LOGISTIC and PHREG procedures of SAS. Cows with VLS 2 had greater incidence of metritis than cows with VLS 0 (69.1 vs. 42.4%), and cows with VLS 1 tended to have greater incidence of metritis than cows with VLS 0 (52.0 vs. 42.4%). Cows with VLS 2 had greater incidence of PVD than cows with VLS 0 (56.5 vs. 43.1%). A lower proportion of cows with VLS 2 than VLS 0 were cyclic by 64 DIM (70.0 vs. 86.8%). A lower proportion of cows with VLS 2 than VLS 0 were pregnant at 60 d after first AI (28.7 vs. 33.6%). Proportion of pregnant cows at 60d after AI tended to be lower for VLS 1 than VLS 0 (28.4 vs. 33.6%). Hazard of pregnancy by 300 DIM was not affected by VLS. Hazard of pregnancy was decreased for cows with metritis, PVD, and anovular cows. In summary, vulvovaginal laceration was associated with uterine disease and cyclicity, which were negatively associated with reproductive performance. Vulvovaginal laceration was recognized as a risk factor for postpartum uterine disease.

Quantifying known and emerging uterine pathogens, and evaluating their association with metritis and fever in dairy cows

Metritis is caused by polymicrobial infection; however, recent metagenomic work challenges the importance of known pathogens such as Escherichia coli and Trueperella pyogenes while identifying potential new pathogens such as Bacteroides pyogenes, Porphyromonas levii and Helcococcus ovis. This study aims to quantify known and emerging uterine pathogens, and to evaluate their association with metritis and fever in dairy cows. Metritis was diagnosed at 6 ± 2 days postpartum, a uterine swab was collected and rectal temperature was measured. 39 cows were classified into three groups: Healthy (n = 14), Metritis without fever (MNoFever; n = 12), and Metritis with fever (MFever; n = 13). Absolute copy number was determined for total bacteria and for 8 potentially pathogenic bacteria using droplet digital PCR. Both MNoFever and MFever cows had higher copy number of total bacteria, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis than Healthy cows. MNoFever and MFever groups were similar. There was no difference among groups in copy number of Escherichia coli, Trueperella pyogenes, and Bacteroides heparinolyticus, and they all had low copy numbers. Our work confirms the importance of some bacteria identified by culture-based studies in the pathogenesis of metritis such as Fusobacterium necrophorum and Prevotella melaninogenica; however, it challenges the importance of others such as Escherichia coli and Trueperella pyogenes at the time of metritis diagnosis. Additionally, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were recognized as emerging pathogens involved in the etiology of metritis. Furthermore, fever was not associated with the total bacterial load or specific bacteria.

Effects of level of dietary cation-anion difference and duration of prepartum feeding on performance and metabolism of dairy cows

The objectives were to evaluate the effects of feeding diets with 2 levels of negative dietary cation-anion differences (DCAD) during the last 42 or 21 d of gestation on performance and metabolism in dairy cows. The hypothesis was that extending feeding from 21 to 42 d and reducing the DCAD from -70 to -180 mEq/kg of dry matter (DM) would not be detrimental to performance. Holstein cows at 230 d of gestation were blocked by parity prepartum (48 entering their second lactation and 66 entering their third or greater lactation) and 305-d milk yield, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial. The 2 levels of DCAD, -70 or -180 mEq/kg of DM, and 2 feeding durations, the last 21 d (short) or the last 42 d (long) prepartum resulted in 4 treatments, short -70 (n = 29), short -180 (n = 29), long -70 (n = 28) and long -180 (n = 28). Cows in the short treatments were fed a diet with DCAD of +110 mEq/kg of DM from -42 to -22 d relative to calving. After calving, cows were fed the same diet and production and disease incidence were evaluated for 42 d in milk, whereas reproduction and survival was evaluated for 305 d in milk. Blood was sampled pre- and postpartum for quantification of metabolites and minerals. Reducing the DCAD linearly decreased prepartum DM intake between -42 and -22 d relative to calving (+110 mEq/kg of DM = 11.5 vs. -70 mEq/kg of DM = 10.7 vs. -180 mEq/kg of DM = 10.2 ± 0.4), and a more acidogenic diet in the last 21 d of the dry period reduced intake by 1.1 kg/d (-70 mEq/kg of DM = 10.8 vs. -180 mEq/kg of DM = 9.7 ± 0.5 kg/d). Cows fed the -180 mEq/kg of DM diet had increased concentrations of ionized Ca in blood on the day of calving (-70 mEq/kg of DM = 1.063 vs. -180 mEq/kg of DM = 1.128 ± 0.020 mM). Extending the duration of feeding the diets with negative DCAD from 21 to 42 d reduced gestation length by 2 d (short = 277.2 vs. long = 275.3 d), milk yield by 2.5 kg/d (short = 40.4 vs. long = 37.9 ± 1.0 kg/d) and tended to increase days open because of reduced pregnancy per artificial insemination (short = 35.0 vs. long = 22.6%). Results suggest that increasing the duration of feeding diets with negative DCAD from 21 to 42 d prepartum might influence milk yield and reproduction of cows in the subsequent lactation, although yields of 3.5% fat- and energy-corrected milk did not differ with treatments. Reducing the DCAD from -70 to -180 mEq/kg of DM induced a more severe metabolic acidosis, increased ionized Ca concentrations prepartum and on the day of calving, and decreased colostrum yield in the first milking, but had no effects on performance in the subsequent lactation. Collectively, these data suggest that extending the feeding of an acidogenic diet beyond 21 d is unnecessary and might be detrimental to dairy cows, and a reduction in the DCAD from -70 to -180 mEq/kg of DM is not needed.