In this prospective cohort study, Holstein cows considered to be at high risk of developing metritis (dystocia, twins, stillbirth, retained placenta, or their combination) were matched with herdmates ...at low risk of developing metritis (normal calving) and monitored daily for rectal temperature and uterine discharge during the first 12 d in milk (DIM). Blood was sampled on d 0, 1, and 3 postpartum for assessment of neutrophil phagocytic and oxidative burst activities. Blood was also sampled at 0, 1, 2, 3, 4, 7, and 12 DIM for determination of serum concentrations of Ca, K, Mg, nonesterified fatty acids, β-hydroxybutyrate, and glucose. On the basis of receiver operator characteristic curves, subclinical hypocalcemia (SCH) was defined as a serum Ca concentration ≤8.59mg/dL in at least 1 sample in the first 3 DIM. The overall incidences of metritis and puerperal metritis were 47.3 and 30%, respectively. Concentration in blood and percentages of neutrophils undergoing phagocytosis and oxidative burst were all reduced in cows with SCH compared with normocalcemic cows. Cows with SCH were at a greater risk of developing fever, metritis, and puerperal metritis compared with normocalcemic cows. Among cows at low risk of developing metritis, those with SCH had a greater incidence of metritis (40.7%) compared with normocalcemic cows (14.3%). Similarly, among cows at high risk of developing metritis, cows with SCH had a greater incidence of metritis (77.8%) compared with normocalcemic cows (20.0%). Cows with SCH had elevated concentrations of nonesterified fatty acids and β-hydroxybutyrate compared with normocalcemic cows. The relative risk of developing metritis decreased by 22% for every 1mg/dL increase in serum Ca. Neither metritis nor SCH influenced the resumption of estrous cyclicity by 38 DIM, but cows with SCH had a reduced pregnancy rate and a longer interval to pregnancy compared with normocalcemic cows. Finally, the population risk to develop uterine diseases attributable to SCH was 66.6% for metritis and 91.3% for puerperal metritis in the present study.
Objectives were to evaluate differences in the uterine and serum metabolomes associated with metritis in dairy cows. Vaginal discharge was evaluated using a Metricheck device (Simcro) at 5, 7, and 11 ...d in milk (DIM; herd 1) or 4, 6, 8, 10, and 12 DIM (herd 2). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 24). Cows with metritis were paired with herdmates without metritis (i.e., clear mucous vaginal discharge or clear lochia with ≤50% of pus) based on DIM and parity (n = 24). Day of metritis diagnosis was considered study d 0. All cows diagnosed with metritis received antimicrobial therapy. The metabolome of uterine lavage collected on d 0 and 5, and serum samples collected on d 0 were evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Normalized data were subjected to multivariate canonical analysis of population using the MultBiplotR and MixOmics packages in R Studio. Univariate analyses including t-test, principal component analyses, partial least squares discriminant analyses, and pathway analyses were conducted using Metaboanalyst. The uterine metabolome differed between cows with and without metritis on d 0. Differences in the uterine metabolome associated with metritis on d 0 were related to the metabolism of butanoate, amino acids (i.e., glycine, serine, threonine, alanine, aspartate, and glutamate), glycolysis and gluconeogenesis, and the tricarboxylic acid cycle. No differences in the serum metabolome were observed between cows diagnosed with metritis and counterparts without metritis on d 0. Similarly, no differences in uterine metabolome were observed between cows with metritis and counterparts not diagnosed with metritis on d 5. These results indicate that the establishment of metritis in dairy cows is associated with local disturbances in amino acid, lipid, and carbohydrate metabolism in the uterus. The lack of differences in the uterine metabolome on d 5 indicates that processes implicated with the disease are reestablished by d 5 after diagnosis and treatment.
The objectives were to characterize the prevalence of periparturient diseases and their effects on reproductive performance of dairy cows in seasonal grazing farms. A total of 957 multiparous cows in ...2 farms (555 in farm A and 402 in farm B) were evaluated and diseases characterized. At calving, dystocia, twin birth, stillbirth, and retained fetal membranes were recorded and grouped as calving problems. On d 7±3 and 14±3 postpartum, cows were evaluated for metritis and on d 28±3 for clinical endometritis based on scoring of the vaginal discharge. From parturition to 30d after artificial insemination (AI), prevalence of mastitis, lameness, and digestive and respiratory problems were recorded. For subclinical diseases, diagnosis was based on blood samples collected from 771 cows and analyzed for concentrations of Ca, nonesterified fatty acids (NEFA), and β-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70mM, subclinical ketosis if the β-hydroxybutyrate concentration was ≥0.96mM, and subclinical hypocalcemia if the Ca concentration was ≤2.14mM. Ovaries were scanned on d 35±3 and 49±3 postpartum for determination of estrous cyclicity. All cows were enrolled in a timed AI program and inseminated on the first day of the breeding season: on average, 86d postpartum. Overall, 37.5% (359/957) of the cows presented at least 1 clinical disease and 59.0% (455/771) had at least 1 subclinical health problem. Prevalence of individual diseases was 8.5% for calving problems, 5.3% for metritis, 15.0% for clinical endometritis, 13.4% for subclinical endometritis, 15.3% for mastitis, 2.5% for respiratory problems, 4.0% for digestive problems, 3.2% for lameness, 20.0% for elevated NEFA concentration, 35.4% for subclinical ketosis, and 43.3% for subclinical hypocalcemia. Clinical and subclinical diseases had additive negative effects on reproduction, delaying resumption of estrous cyclicity and reducing pregnancy per AI (P/AI). Occurrence of multiple diseases further reduced reproductive efficiency compared with a single disease. Individually, subclinical hypocalcemia, elevated NEFA concentration, metritis, and respiratory and digestive problems reduced estrous cyclicity by d 49 postpartum. Elevated NEFA concentration, calving problem, metritis, clinical and subclinical endometritis, and digestive problems reduced P/AI on d 65 after AI. Moreover, calving problems and clinical endometritis increased the risk of pregnancy loss between gestation d 30 and 65. Serum concentrations of Ca and NEFA were negatively correlated, and both were associated with prevalence of uterine diseases. In conclusion, periparturient diseases were highly prevalent in seasonally calving grazing dairies and affected cows had delayed resumption of estrous cyclicity, reduced P/AI, and increased risk of pregnancy loss.
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.
Use of assisted reproduction techniques (ART) such as ovum pick-up for in vitro embryo production (OPU-IVP) and multiple ovulation for embryo transfer (MOET) in livestock has increased in recent ...years. Objectives of this retrospective cohort study were to evaluate the association between ART and reproductive performance of donor dairy heifers and cows until third lactation. Holsteins donors subjected to MOET (n = 1087) or OPU-IVP (n = 551) were matched with non-donor females (n = 4112) based on date of birth allowing for a maximal difference of 10 days. Binary variables were analyzed using multivariable logistic regression whereas time to first breeding and time to pregnancy were analyzed using multivariable Cox’s proportional hazard models and Kaplan-Meier survival curves. Contrasts were built to assess the association between donor status (non-donor vs. MOET + OPU-IVP), type of ART (MOET vs. OPU-IVP) and number of OPU-IVP procedures. Non-donor heifers had greater hazards of first breeding, pregnancy, and first calving compared with donor counterparts. Proportion of pregnant heifers after first breeding was reduced for donors compared with non-donors, which was primarily explained by a reduced proportion of OPU-IVP donors that became pregnant. Although proportion of pregnant cows after first breeding was not associated with ART group during first lactation, hazards of first breeding and pregnancy relative to the end of voluntary wait period were reduced for OPU-IVP donors. During second and third lactations, hazard of first breeding was greater for non-donors compared with donor counterparts. However, proportion of pregnant cows after first breeding and pregnancy hazard were not associated with ART groups. Culling was greater for non-donors compared with donors across all lactations. However, proportion cows culled during first lactation that was attributed to reproductive causes tended to be greater for donor compared with non-donor cows. Assisted reproduction techniques were associated with reduced reproductive performance in dairy heifers and cows but did not increase overall culling.
•Embryo donors had reduced hazard of first service and pregnancy linked to the time required for super stimulation protocols.•Ovum pick-up was associated with reduced pregnancy hazard in heifers and first lactation cows.•Fertility losses observed in Holstein donors were not associated with increased culling.
Two experiments evaluated the influence of follicular wave at artificial insemination (AI) on fertility of dairy cows. In experiment 1, data from 5,607 lactating cows enrolled in estrous and ...ovulation synchronization programs for AI were evaluated. Cows’ blood was analyzed for progesterone 7 to 14 d apart, with the second sample collected on the day of the first GnRH (GnRH1) of the synchronization protocol. Cows were classified as cyclic if progesterone was ≥1 ng/mL in at least 1 of the 2 samples and as anovular if both samples were <1 ng/mL. Cyclic cows were categorized as low (CLOW;<1 ng/mL) or high (CHIGH; ≥ 1 ng/mL) progesterone on the day of GnRH1, which would result in ovulation of the dominant follicle of the first (FW) and second (SW) follicular waves, respectively, at AI. Pregnancy per AI (P/AI) was determined 30 and 53 d after AI. In experiment 2, 220 cyclic Holstein cows received 2 injections of PGF2α administered 14 d apart. The Ovsynch protocol (d 0 GnRH, d 7 PGF2α, d 9 GnRH, d 9.5 timed AI) was initiated either 3 or 10 d after the second PGF2α of the presynchronization to result in insemination to the FW or SW dominant follicles. Blood was analyzed for progesterone and ovaries were scanned to determine ovulatory responses and follicle diameter. Pregnancy was determined on d 32 and 67 after timed AI. In experiment 1, P/AI on d 30 was greater for CHIGH cows than for CLOW and anovular cows (43.0, 31.3, and 29.7%, respectively), but because of pregnancy loss, P/AI on d 53 was lowest for anovular cows. Proportions of cows with short reinsemination intervals differed among groups and were 7.1, 15.7, and 11.9% for CHIGH, CLOW, and anovular cows, respectively. Pregnancy loss was greater for anovular cows than for CLOW cows (15.0 vs. 10.0%) and was intermediate for CHIGH cows (13.5%). In experiment 2, 9.8 and 97.2% of the FW and SW cows, respectively, had progesterone ≥1 ng/mL at GnRH1. Concentrations of progesterone at the GnRH1 and PGF2α injections of the Ovsynch protocol were greater for SW cows than FW cows. Pregnancy per AI was greater for SW cows than for FW cows (41.7 vs. 30.4%) despite less ovulation to GnRH1 in SW cows than in FW cows (78.7 vs. 88.4%). Collectively, these data indicate that follicular wave of the ovulatory follicle and not cyclic status caused the greatest reduction in P/AI in dairy cows. Whether the culprit is the follicle itself or the hormonal milieu characteristic of the first follicular wave and the early stage of the estrous cycle remains to be elucidated. Synchronization programs that induced ovulation of the FW follicle at AI reduced P/AI in lactating dairy cows, and ovulation of the FW follicle, or development of the ovulatory follicle under low progesterone concentrations, or both, might be mechanisms for reduced fertility in anovular cows.
Two experiments were conducted to evaluate the effects of the timing of artificial insemination (AI) and incorporation of the Slo3 K+ channel blocker 4-(4-chlorophenyl)butyl-diethyl-heptylammonium to ...semen extender (CSE) on pregnancy per AI (P/AI) and pregnancy loss in dairy heifers. In experiment 1, Holstein heifers were subjected to the 5-d CIDR-Synch protocol: d −8 GnRH and controlled internal drug-release device (CIDR); d −3 PGF2α and CIDR removal; d −2 PGF2α; d 0 GnRH) and assigned randomly to receive timed AI with control semen on d 0 (72-CON; n = 104), control semen on d −1 (48-CON; n = 100), or CSE-treated semen on d −1 (48-CSE; n = 98). Heifers were fitted with collar-mounted automated estrus detection devices to monitor physical activity and rumination. In experiment 2, Holstein heifers were subjected to the 5-d CIDR-Synch protocol and received a mount detection patch at the first PGF2α injection. Heifers detected in estrus before d 0 were inseminated on the same day, whereas those not detected in estrus received the second GnRH injection and timed AI on d 0. Heifers were assigned randomly to receive AI with control (AI-CON; n = 148) or CSE-treated semen (AI-CSE; n = 110). Four bulls with proven fertility were used in both experiments, and ejaculates from each sire were divided and processed as CON or CSE. Pregnancy was diagnosed by transrectal ultrasonography at 29 and 54 d after AI. Data were analyzed by logistic regression, and statistical models included the fixed effects of treatment and enrollment week. In experiment 1, orthogonal contrasts were built to assess the effects of day of AI (72-CON vs. 48-CON + 48-CSE) and treatment of semen with CSE (48-CON vs. 48-CSE). Pregnancy per AI on d 29 (72-CON = 60.8, 48-CON = 35.2, 48-CSE = 39.8%) and d 54 (72-CON = 58.2, 48-CON = 31.6, 48-CSE = 36.2%) was greater for heifers inseminated on d 0 compared with d −1. However, no effect of semen extender on P/AI was observed in heifers inseminated on d −1. In experiment 2, P/AI tended to be greater for AI-CSE than for AI-CON on d 29 (58.6 vs. 47.3%) and d 54 after AI (55.6 vs. 43.7%). Advancing AI by 24 h decreased the likelihood of pregnancy, and use of CSE was unable to overcome the expected asynchrony between insemination and ovulation. Nevertheless, incorporation of CSE in semen processing tended to improve P/AI when heifers received AI upon detected estrus or timed AI concurrently with the final GnRH of the 5-d CIDR-Synch protocol.
Resumption of ovulation after parturition is a coordinated process that involves recoupling of the GH/insulin-like growth factor 1 axis in the liver, increase in follicular development and ...steroidogenesis, and removal of negative feedback from estradiol in the hypothalamus. Infectious diseases and metabolic disorders associated with extensive negative energy balance during early lactation disrupt this pathway and delay first ovulation postpartum. Extended periods of anovulation postpartum exert long-lasting effects on fertility in dairy cows including the lack of spontaneous estrus, reduced pregnancy per artificial insemination (P/AI), and increased risk of pregnancy loss. Concentrations of progesterone in anovular cows subjected to synchronized programs for AI are insufficient to optimize follicular maturation, oocyte competence, and subsequent fertility to AI. Ovulation of first wave follicles, which develop under low concentrations of progesterone, reduces embryo quality in the first week after fertilization and P/AI in dairy cows. Although the specific mechanisms by which anovulation and low concentrations of progesterone impair oocyte quality have not been defined, studies with persistent follicles support the involvement of premature resumption of meiosis and degradation of maternal RNA. Suboptimal concentrations of progesterone before ovulation also increase the synthesis of PGF2α in response to oxytocin during the subsequent estrous cycle, which explains the greater incidence of short luteal phases after the first AI postpartum in anovular cows compared with estrous cyclic herd mates. It is suggested that increased spontaneous luteolysis early in the estrous cycle is one of the mechanisms that contributes to early embryonic losses in anovular cows. Anovulation also leads to major shifts in gene expression in elongated conceptuses during preimplantation stages of pregnancy. Transcripts involved with control of energy metabolism and DNA repair were downregulated, whereas genes linked to apoptosis and autophagy were upregulated in Day 15 conceptuses collected from anovular cows compared with estrous cyclic counterparts. Similar changes in conceptus transcriptome were not observed in estrous cyclic cows induced to ovulate follicles that grew under low and high concentrations of progesterone, indicating an effect of anovulation on embryonic development that is not mediated solely by progesterone concentrations before ovulation. Finally, risk factors for anovulation have direct effects on embryo development and uterine receptivity to pregnancy that complement those determined by insufficient concentrations of progesterone during follicular growth. One approach to minimize the impact of anovulation on fertility is supplementation with progesterone during recruitment, selection and final stages of development of the preovulatory follicle. It is suggested that a minimum of 2.0 ng/mL of progesterone is needed during growth of the preovulatory follicle to achieve P/AI similar to that of cows growing the preovulatory follicle during diestrus.
Objectives were to investigate 2 intervals from induction of ovulation to artificial insemination (AI) and the effect of supplemental progesterone for resynchronization on fertility of lactating ...dairy cows subjected to a 5-d timed AI program. In experiment 1, 1,227 Holstein cows had their estrous cycles presynchronized with 2 injections of PGF2α at 46 and 60 d in milk (DIM). The timed AI protocols were initiated with GnRH at 72 DIM, followed by 2 injections of PGF2α at 77 and 78 DIM and a second injection of GnRH at either 56 (OVS56) or 72h (COS72) after the first PGF2α of the timed AI protocols. All cows were time-inseminated at 72h after the first PGF2α injection. Pregnancy was diagnosed on d 32 and 60 after AI. In experiment 2, 675 nonpregnant Holstein cows had their estrous cycles resynchronized starting at 34 d after the first AI. Cows received the OVS56 with (RCIDR) or without (RCON) supplemental progesterone, as an intravaginal insert, from the first GnRH to the first PGF2α. Pregnancy diagnoses were performed on d 32 and 60 after AI. During experiment 2, subsets of cows had their ovaries scanned by ultrasonography at the first GnRH, the first PGF2α, and second GnRH injections of the protocol. Blood was sampled on the day of AI and 7 d later, and concentrations of progesterone were determined in plasma. Cows were considered to have a synchronized ovulation if they had progesterone <1 and >2.26ng/mL on the day of AI and 7 d later, respectively, and if no ovulation was detected between the first PGF2α and second GnRH injections during resynchronization. In experiment 1, the proportion of cows detected in estrus at AI was greater for COS72 than OVS56 (40.6 vs. 32.4%). Pregnancy per AI (P/AI) did not differ between OVS56 (46.4%) and COS72 (45.5%). In experiment 2, cows supplemented with progesterone had greater P/AI compared with unsupplemented cows (51.3 vs. 43.1%). Premature ovulation tended to be greater for RCON than RCIDR cows (7.5 vs. 3.6%), although synchronization of the estrous cycle after timed AI was similar between treatments. Timing of induction of ovulation with GnRH relative to insemination did not affect P/AI of dairy cows enrolled in a 5-d timed AI program. Furthermore, during resynchronization starting on d 34 after the first AI, supplementation with progesterone improved P/AI in cows subjected to the 5-d timed AI protocol.
The objectives were to study the effects of induced subclinical hypocalcemia SCH, blood ionized Ca (iCa2+) <1.0mM, without recumbency on physiological responses and function of immune cells in dairy ...cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43g of oral Ca, as Ca sulfate and Ca chloride, at −1 and 11h) or an induced SCH SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa2+ chelator, intravenous infusion treatment for 24h, using a crossover design. The sequence of treatments was either NC–SCHI or SCHI–NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa2+ <1.0mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, β-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa2+ in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa2+ concentrations throughout the 24-h treatment period (0.77±0.01 vs. 1.26±0.01mM iCa2+). Induction of SCH reduced dry matter intake on the day of infusion (5.3±0.8 vs. 9.1±0.8kg/d) and rumen contractions (1.9±0.2 vs. 2.7±0.2 contractions/2min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44±0.23 vs. 2.32±0.23ng/mL) evident between 6 and 18h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40±0.04 vs. 4.17±0.04mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110±0.019 vs. 0.061±0.014mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa2+ after stimulation with ionomycin (9.9±1.0 vs. 13.6±1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1±2.1 vs. 29.3±2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1±1.7 vs. 24.2±1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.
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