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.

Association of dry matter intake and energy balance prepartum and postpartum with health disorders postpartum: Part II. Ketosis and clinical mastitis.

The main objective of this study was to determine the association of dry matter intake as percentage of body weight (DMI%BW) and energy balance (EB) prepartum (-21 d relative to parturition) and postpartum (28 d) with ketosis (n = 189) and clinical mastitis (n = 79). For this, DMI%BW and EB were the independent variables and ketosis and clinical mastitis were the dependent variables. A secondary objective was to evaluate prepartum DMI%BW and EB as predictors of ketosis and clinical mastitis. For this, ketosis and clinical mastitis were the independent variables and DMI%BW and EB were the dependent variables. Data from 476 cows from 9 experiments were compiled. Clinical mastitis was diagnosed if milk from 1 or more quarters was abnormal in color, viscosity, or consistency, with or without accompanying heat, pain, redness, or swelling of the quarter or generalized illness, during the first 28 d postpartum. Ketosis was defined as the presence of acetoacetate in urine that resulted in any color change [5 mg/dL (trace) or higher] in the urine test strip (Ketostix, Bayer, Leverkusen, Germany). Cows that developed ketosis had lesser DMI%BW and lesser EB on d -5, -3, -2, and -1 than cows without ketosis. Each 0.1-percentage point decrease in the average DMI%BW and each 1-Mcal decrease in the average of EB in the last 3 d prepartum increased the odds of having ketosis by 8 and 5%, respectively. Cut-offs for DMI%BW and EB during the last 3 d prepartum to predict ketosis were established and were ≤1.5%/d and ≤1.1 Mcal/d, respectively. Cows that developed ketosis had lesser postpartum DMI%BW and EB and greater energy-corrected milk (ECM) than cows without ketosis. Cows that developed clinical mastitis had lesser DMI%BW but similar prepartum EB compared with cows without clinical mastitis. Each 0.1-percentage point decrease in the average DMI%BW and each 1-Mcal decrease in the average EB in the last 3 d prepartum increased the odds of having clinical mastitis by 10 and 8%, respectively. The average DMI%BW and EB during the last 3 d prepartum produced significant cut-offs to predict clinical mastitis postpartum, which were ≤1.2%/d and ≤1.0 Mcal/d, respectively. Cows that developed clinical mastitis had lesser postpartum DMI%BW from d 3 to 15 and on d 17; greater EB on d 18, from d 21 to 23, and on d 26; and lesser ECM. The main limitation in this study is that the time-order of disease relative to DMI%BW and ECM is inconsistent such that postpartum outcomes were measured before and after disease, which was diagnosed at variable intervals after calving. In summary, measures of prepartum DMI were associated with and were predictors of ketosis and clinical mastitis postpartum, although the effect sizes were small.

Association of dry matter intake and energy balance prepartum and postpartum with health disorders postpartum: Part I. Calving disorders and metritis.

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

Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.

The objectives of this experiment were to evaluate the effect of feeding a culture of Saccharomyces cerevisiae on rumen metabolism and digestibility when cows are fed diets varying in starch content. Four lactating Holstein cows were assigned to a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Treatments were low starch (LS; 23% of diet DM) and no yeast culture (YC; LS-control), LS and 15 g of YC/d (LS-YC), high starch (HS; 29% of diet DM) and no YC (HS-control), and HS and 15 g of YC/d (HS-YC). Periods lasted 28 d, with the last 9 d for data collection. Days 20 to 24 were used to determine production, nutrient flow, and digestibility. On d 25, 3 kg of corn grain DM was placed in the rumen 1 h before the morning feeding, and yields of milk and milk components were measured after the challenge. Blood was sampled -1, 3, 7, and 11 h relative to the morning feeding on d 24 and 25. Rumen pH was measured continuously on d 24 and 25. Rumen papillae were collected on d 24 and 28 to quantify mRNA expression of select genes. Supplementing YC increased yields of milk (26.3 vs. 29.6 kg/d), energy-corrected milk (ECM; 26.5 vs. 30.3 kg/d), fat (0.94 vs. 1.08 kg/d), true protein (0.84 vs. 0.96 kg/d), and ECM/dry matter intake (1.15 vs. 1.30) compared with the control but did not affect dry matter intake (22.6 vs. 22.9 kg/d). Cows fed HS had increased milk true protein percentage (3.18 vs. 3.31%) and yield (0.87 vs. 0.94 kg/d) compared with cows fed LS. Feeding HS-YC increased the proportion of dietary N incorporated into milk true protein from 24.9% in the other 3 treatments to 29.6%. Feeding HS increased the concentration of propionate (21.7 vs. 32.3 mM) and reduced that of NH3-N (8.3 vs. 6.7 mg/dL) in rumen fluid compared with the control, and combining HS with YC in HS-YC tended to increase microbial N synthesis compared with LS-YC (275 vs. 322 g/d). Supplementing YC to cows fed HS reduced plasma haptoglobin and rumen lactate concentrations, increased mean rumen pH, reduced the time with pH <6.0, and prevented the decrease in rumen neutral detergent fiber digestion caused by HS. Cows fed HS had less total-tract digestion of organic matter (73.9 vs. 72.4%) because of reduced acid detergent fiber (57.6 vs. 51.7%) and neutral detergent fiber (60.9 vs. 56.7%) digestibility. Production performance after the challenge was similar to that before the challenge, and YC improved yield of ECM. After the challenge, supplementing YC tended to reduce rumen lactate concentration compared with the control and reduced haptoglobin in cows fed HS. Feeding HS but not YC increased expression in rumen papillae of genes for receptors (FFAR2 and FFAR3) and transporter (SLC16A3) of short-chain fatty acids but did not affect genes involved in transport of Na+/H+ or water or in inflammatory response. Supplementing YC to dairy cows improved lactation performance in diets containing low or high starch, and mechanisms might be partially attributed to improvements in rumen pH, digestion of fiber, microbial N synthesis, and reduction in acute phase response.

Effects of supplementing yeast culture to diets differing in starch content on performance and feeding behavior of dairy cows.

The objectives were to evaluate the effects of a culture of Saccharomyces cerevisiae (YC) on lactation performance of cows fed diets differing in starch content. Fifty-six Holstein cows at 42 d postpartum were blocked by parity and milk production and randomly assigned to 1 of 4 treatments, low starch (23% diet DM) and no YC (LS-control), low starch and 15 g/d of YC (LS-YC), high starch (29% diet DM) and no YC (HS-control), and high starch and 15 g/d of YC (HS-YC). The experiment lasted 14 wk. Blood was sampled twice weekly during the first 5 wk in the experiment. Feeding behavior was evaluated in 2 consecutive days when cows were 33 d in the experiment. On d 92 in the experiment, cows were challenged with 3 kg of corn grain DM immediately before the morning feeding. Blood was sampled in the first 12 h after the challenge. Rumen fluid was collected 5 h after the challenge, and pH, ammonia N, short-chain fatty acids, and lactate concentrations were quantified. Lactation performance was measured daily before and after the challenge. Supplementation with YC increased yields of 3.5% fat-corrected milk and energy-corrected milk by 2.2 and 2.0 kg/d, and the increments were observed in both low- and high-starch diets. Feeding HS tended to decrease milk fat content (LS = 3.88 vs. HS = 3.73%), but increased concentration (LS = 2.87 vs. HS = 3.00%) and yield (LS = 1.11 vs. HS = 1.20 kg/d) of milk true protein. Feeding YC increased yields of fat and true protein in milk by 100 and 60 g/d. Energy balance, body weight, and feed efficiency did not differ with treatments. Feeding HS reduced eating time (LS = 177 vs. HS = 159 min/12 h) and intermeal interval (LS = 103 vs. HS = 82 min), but tended to increase eating rate (LS = 139 vs. HS = 150 g/min). Interactions were detected between level of starch and YC for ruminating time, meal duration, and meal size because within LS, feeding YC increased ruminating time 23 min/12 h, but reduced meal duration 6 min/meal and meal size 0.7 kg/meal. Concentrations of glucose in plasma increased (LS = 62.1 vs. HS = 63.8 mg/dL), whereas those of urea N decreased (LS = 10.1 vs. HS = 9.4 mg/dL) with feeding HS compared with LS in the first 5 wk in the experiment, and the same responses were observed after the challenge with corn grain. After the challenge, rumen pH was less and short-chain fatty acid concentrations were greater in cows fed HS compared with those fed LS; however, supplementing YC to high-starch diets increased rumen pH (HS-control = 5.72 vs. HS-YC = 6.12) and reduced concentrations of lactate in rumen fluid (HS-control = 7.72 vs. HS-YC = 1.33 mM) and haptoglobin in plasma 28%. Feeding YC improved lactation performance irrespective of the level of dietary starch and reduced the risk of subacute rumen acidosis induced by a grain challenge when cows were fed a high-starch ration.

Effects of altering the ratio of dietary n-6 to n-3 fatty acids on spontaneous luteolysis in lactating dairy cows.

Objectives were to evaluate the effects of altering the dietary ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids on the profile of fatty acids and expression of genes related to the prostaglandin biosynthesis on endometrial tissue, uterine secretion of PGF2α, and timing of spontaneous luteolysis in dairy cows. Multiparous lactating Holstein cows (n = 45) were blocked based on milk yield and, within each block, assigned randomly to 1 of 3 dietary treatments at 14 d postpartum for 90 d. Diets were supplemented with a mixture of Ca salts of fish, safflower, and palm oils to create 3 different ratios of n-6 to n-3 fatty acids, namely R4, R5, and R6, which resulted in 3.9, 4.9, and 5.9 parts of n-6 to 1 part of n-3 fatty acids, respectively. Blood was sampled every 2 h from d 16 to 23 of the estrous cycle and assayed for concentrations of progesterone and the PGF2α metabolite 13,14-dihydro-15-keto-PGF2α (PGFM). In a subsequent estrous cycle, endometrial tissue was collected for biopsy on d 8 and endometrial fatty acids profile and gene expression were quantified. The proportion of arachidonic acid of the endometrial fatty acids increased as the dietary ratio n-6 to n-3 fatty acids increased (R4 = 9.05, R5 = 11.64, and R6 = 13.41%). On the other hand, proportions of eicosapentaenoic (R4 = 2.85, R5 = 2.14, and R6 = 2.02%) and docosahexaenoic (R4 = 3.30, R5 = 1.57, and R6 = 1.08%) decreased as the ratio of n-6 to n-3 fatty acids in the diet increased. Increasing the ratio of dietary n-6 to n-3 fatty acids increased mRNA expression of estrogen receptor 1, oxytocin receptor, cyclooxygenase 2, prostaglandin E and F synthases, and steroidogenic acute regulatory protein in endometrium, but decreased expression of peroxisome proliferator-activated receptor gamma and insulin-like growth factor-1. The changes in endometrium gene expression caused by dietary treatments were associated with changes in the ratio of n-6 to n-3 fatty acids in the endometrium. As the ratio increased from R4 to R6, the number of PGFM pulses (R4 = 5.6, R5 = 4.3, and R6 = 3.8 ± 0.6 pulses; least squares means ± standard error of the means) decreased, but the amplitude of the greatest PGFM pulse increased (R4 = 226, R5 = 267, and R6 = 369 ± 38 pg/mL). Luteolysis by d 23 of the estrous cycle was observed in 79.6% of the cows (R4 = 11/14; R5 = 13/15; and R6 = 11/15) and day of spontaneous luteolysis did not differ among treatments (R4 = 20.8; R5 = 21.1; and R6 = 21.0 ± 0.4). Three pulses of PGFM was the best predictor of luteolysis in dairy cows. Collectively, supplying the same quantity of fatty acids in the diet of lactating dairy cows, but altering the ratio of n-6 to n-3 fatty acids, influenced the endometrial fatty acids profile and gene expression and altered the pattern of prostaglandin synthesis; however, the changes were not sufficient to alter the length of the estrous cycle.

Supplementation of essential fatty acids to Holstein calves during late uterine life and first month of life alters hepatic fatty acid profile and gene expression.

Linoleic acid is an essential dietary fatty acid (FA). However, how the supplementation of linoleic acid during uterine and early life may modify the FA profile and transcriptome regulation of the liver, and performance of preweaned dairy calves is unknown. Our objective was to evaluate the effect of supplementation of essential FA to Holstein calves during late uterine and early life on their hepatic FA profile and global gene expression at 30 d of age. During the last 8 wk of pregnancy, Holstein cattle (n=96) were fed either no fat supplement (control), a saturated FA supplement enriched with C18:0, or an unsaturated FA supplement enriched with linoleic acid. Male calves (n=40) born from these dams were fed a milk replacer (MR) with either low (LLA) or high linoleic acid (HLA) concentration as the sole feedstuff during the first 30 d. Liver biopsy was performed at 30 d of age, and microarray analysis was performed on 18 liver samples. Total concentration of FA in liver were greater in calves fed LLA compared with those fed HLA MR (8.2 vs. 7.1%), but plasma concentrations of total FA did not differ due to MR diets. The FA profiles of plasma and liver of calves were affected differently by the prepartum diets. Specifically, the FA profile in liver was affected moderately by the feeding of fat prepartum, but the profiles did not differ due to the type of FA fed prepartum. The type of MR fed during the first 30 d of life had major effects on both plasma and liver FA profiles, resembling the type of fat fed. Plasma and liver of calves fed LLA MR had greater percentage of medium-chain FA (C12:0 and C14:0), whereas plasma and liver from calves fed HLA MR had greater percentages of linoleic and α-linolenic acids. Dams fed fat or a specific type of FA modified the expression of some genes in liver of calves, particularly those genes involved in biological functions and pathways related to upregulation of lipid metabolism and downregulation of inflammatory responses. Feeding HLA instead of LLA MR modified the expression of hepatic genes, including genes predicted to decrease infections and to increase lipid utilization and protein synthesis. Research evaluating the effect of FA supplementation during uterine and neonatal life on the future productivity of the neonate is warranted.

Conceptus development and transcriptome at preimplantation stages in lactating dairy cows of distinct genetic groups and estrous cyclic statuses.

The objectives were to compare development and transcriptome of preimplantation conceptuses 15 d after synchronized ovulation and artificial insemination (AI) according to the genetic background of the cow and estrous cyclicity at the initiation of the synchronization program. On d 39±3 postpartum, Holstein cows that were anovular (HA; n=10), Holstein cows that were estrous cyclic (HC; n=25), and Jersey/Holstein crossbred cows that were estrous cyclic (CC; n=25) were randomly selected in a grazing herd and subjected to the Ovsynch protocol. All cows were inseminated on d 49±3 postpartum, which was considered study d 0. Blood was sampled and analyzed for concentrations of progesterone, estradiol, insulin, and insulin-like growth factor 1 (IGF-1) on study d -10, -3, -1, 7, and 15 relative to AI. On study d 15, uteri were flushed and recovered fluid had IFN-τ concentrations measured and subjected to metabolomic analysis. Morphology of the recovered conceptuses was evaluated, and mRNA was extracted and subjected to transcriptome microarray analysis. Compared with HC, CC presented greater concentrations of progesterone and estradiol in plasma, with corpora lutea and preovulatory follicles of similar size. Conceptuses from CC were larger, tended to secrete greater amounts of IFN-τ, and had greater transcript expression of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor that coordinates lipid metabolism and elongation at preimplantation development. In addition, pregnant CC had greater concentrations of anandamide in the uterine flush, which might be important for elongation of the conceptus and early implantation. Conceptuses from HA were also longer and secreted greater amounts of IFN-τ than conceptuses from HC, likely because of the distinct progesterone profiles before and after AI. Nonetheless, anovular cows had reduced concentrations of IGF-1 in plasma, and their conceptuses presented remarkable transcriptomic differences. Some of the altered transcripts suggest that conceptus cells from anovular cows might be under greater cellular stress and presented markers suggesting increased apoptosis and autophagy, which could lead to increased mortality after d 15 of development. Estrous cyclicity had more impact on transcriptome of bovine conceptus than genetic background, and the developmental changes observed during the preimplantation period might be linked to differences in fertility among groups.

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.

Increasing intake of essential fatty acids from milk replacer benefits performance, immune responses, and health of preweaned Holstein calves.

The objective was to evaluate the effect of feeding increasing amounts of essential fatty acids (FA) in milk replacer (MR) during the first 60 d of life on growth, health, and immunity of Holstein calves. Calves were born from dams fed low concentrations of total and essential FA during the lasT2 mo of pregnancy. Newborn calves were blocked by sex and parity of the dam and assigned randomly to receive 1 of 4 MR treatments (T). Hydrogenated coconut oil and soybean oil were mixed with emulsifier and commercial MR powder to prepare the following 4 MR containing 0.119 and 0.007 (T1), 0.187 and 0.017 (T2), 0.321 and 0.036 (T3), and 0.593 and 0.076 (T4) g of intake per kg of metabolic body weight (BW(0.75)) of linoleic acid and α-linolenic acid, respectively. At 30 d of life, concentrations of essential FA (linoleic acid and α-linolenic acid) in liver increased, whereas concentrations of C12:0, C14:0, C16:0, and C20:3n-9 decreased linearly with increasing intake of essential FA. Body weight gain and feed efficiency were optimized when male calves consumed T2, whereas gain by female calves tended to increase linearly with increasing intake of essential FA during the first 30 d of age. However, these responses to treatment were not maintained after initiation of concentrate feeding at 31 d of life. Over the 60-d preweaning period, wither and hip heights were improved in both sexes as intake of essential FA increased up to T3. Some measures of health and immunity were affected by replacing some coconut oil with soybean oil. Severity of diarrhea tended to decrease linearly; plasma concentrations of haptoglobin during diarrhea were lower in T2, T3, and T4; phagocytosis by blood neutrophils tended to peak for calves fed T2; in vitro proliferation of stimulated blood lymphocytes was greater for calves fed T2; in vitro stimulated blood cells produced more IFN-γ (up to T3 for males and T2 for females), concentrations of serum IgG against ovalbumin injections were increased in males fed T2 or T3; and skin-fold thickness increased in response to an intradermal antigen injection of all calves fed up to T4. Across sex and under the conditions of the present study, mean daily intakes of linoleic acid between 3 to 5g/d and intakes of α-linolenic acid between 0.3 and 0.6g/d during the first 30 d of life promoted growth of preweaned Holstein calves, possibly by improving their immune status.

Effects of altering the ratio of dietary n-6 to n-3 fatty acids on performance and inflammatory responses to a lipopolysaccharide challenge in lactating Holstein cows.

The study was designed to evaluate the effects of altering the ratio between n-6 and n-3 fatty acids (FA) in the diet and the intake of these FA by lactating dairy cows on lactation performance and inflammatory acute phase responses to a challenge with lipopolysaccharide (LPS). Multiparous Holstein cows (n=45) were blocked based on milk yield from d 6 to d 10 postpartum and, within each block, assigned randomly to 1 of 3 dietary treatments at 14d postpartum; treatments lasted for 90d. Diets were supplemented with a mixture of Ca salts of fish, safflower, and palm oils to create 3 different ratios of n-6 to n-3 FA; namely, 3.9, 4.9, or 5.9 parts of n-6 to 1 part of n-3 FA (R4, R5, and R6, respectively). During the first 5 wk of the study, blood was sampled weekly and analyzed for concentrations of metabolites and hormones. On d 75 postpartum, cows received an infusion of 10µg of LPS into one quarter of the mammary gland to evaluate inflammatory acute phase responses. Altering the ratio of dietary n-6 to n-3 FA was reflected in changes in the FA composition of plasma and milk fat. Reducing the ratio of n-6 to n-3 FA from R6 to R4 increased dry matter intake (24.7, 24.6, and 26.1±0.5kg/d for R6, R5, and R4, respectively), with concurrent increases in yields of 3.5% fat-corrected milk (43.4, 45.4, and 48.0±0.8kg/d), milk fat (1.53, 1.60, and 1.71±0.03kg/d), milk true protein (1.24, 1.28, and 1.32±0.02kg/d), and milk lactose (2.12, 2.19, and 2.29±0.04kg/d). After the LPS challenge, concentrations of IL-6 in plasma increased as the ratio of n-6 to n-3 FA increased (112.5, 353.4, and 365.1±86.6pg/mL for R4, R5, and R6, respectively). Elevations of body temperature and somatic cell count were greater for cows fed R5 compared with those fed R4 or R6 (41.3, 40.8, and 40.8±0.2°C; 4.33, 3.68, and 3.58±0.25×10(6)/mL, for R5, R4, and R6, respectively). Haptoglobin concentration was greatest at 24h after LPS challenge for cows fed R6. Phagocytosis and oxidative burst by neutrophils collected from circulation were unaffected by dietary treatment in the first 48h after intramammary LPS infusion. In conclusion, supplying the same quantity of FA in the diet of early lactation dairy cows but altering the ratio of the polyunsaturated FA of the n-6 to n-3 families influenced lactation performance and inflammatory responses to an LPS challenge.

Plasma anti-Müllerian hormone in adult dairy cows and associations with fertility.

The objectives of this study were to identify factors associated with concentrations of anti-Müllerian hormone (AMH) in plasma of dairy cows and to investigate the relationships between plasma AMH and fertility responses during a 100-d breeding season. Lactating cows, 1,237 in 2 seasonally calving herds, had estrous cycles presynchronized and were enrolled in a timed artificial insemination (AI) protocol. All cows were inseminated on the first day of breeding season, considered study d 0. Blood was sampled on d -8 and analyzed for concentrations of AMH and progesterone. From d 19 to 35, detection of estrus was performed daily and cows detected in estrus were reinseminated. On d 36, bulls were placed with cows for 65 d of natural service breeding. Factors identified to be associated with concentrations of AMH in plasma were breed of the cow and lactation number. Concentrations of AMH were greater for Jerseys, followed by crossbreds, and then Holsteins. Cows on lactations 2 and 3 had greater concentrations of AMH than those on lactations 1 and ≥ 4. Although pregnancy per AI at the timed AI was not associated with concentrations of AMH, cows with low AMH had greater detection of estrus at timed AI, and the latter benefited pregnancy per AI, particularly in cows that had low progesterone at the beginning of the synchronization protocol. Pregnancy loss between gestation d 30 and 65 was greater in cows with low AMH compared with those with intermediate or high AMH. Return to estrus in cows that failed to become pregnant from the timed AI was not associated with AMH, but pregnancy rate in cows bred on estrus (reinsemination + natural service) was associated positively with AMH. In conclusion, breed and lactation number were identified to be associated with concentrations of AMH in plasma. Concentration of AMH was associated positively with maintenance of pregnancy at the first postpartum AI and with pregnancy rate in cows inseminated after detection of spontaneous estrus. Synchronization of ovulation might override positive associations between AMH and fertility.

Effect of supplementing fat to pregnant nonlactating cows on colostral fatty acid profile and passive immunity of the newborn calf.

The objectives were to evaluate the effect of supplementing saturated or unsaturated long-chain fatty acids (FA) to nulliparous and parous Holstein animals (n=78) during late gestation on FA profile of colostrum and plasma of newborn calves and on production and absorption of IgG. The saturated FA supplement (SAT) was enriched in C18:0 and the unsaturated FA supplement (ESS) was enriched in the essential FA C18:2n-6. Fatty acids were supplemented at 1.7% of dietary dry matter to low-FA diets (1.9% of dietary dry matter) during the last 8 wk of gestation. Calves were fed 4 L of colostrum within 2h of birth from their own dam or from a dam fed the same treatment. Feeding fat did not affect prepartum dry matter intake, body weight change, or gestation length. Parous but not nulliparous dams tended to give birth to heavier calves if fed fat prepartum. Parous dams were less able to synthesize essential FA derivatives, as evidenced by lower desaturase indices, compared with nulliparous dams, suggesting a greater need for essential FA supplementation. The FA profile of colostrum was modified to a greater degree by prepartum fat feeding than was that of neonatal calf plasma. The placental transfer and synthesis of elongated n-3 FA (C20:5, C22:5, and C22:6) were reduced, whereas the n-6 FA (C18:2, C18:3, and C20:3) were increased in plasma of calves born from dams fed ESS rather than SAT. Supplementing unsaturated FA prepartum resulted in elevated concentrations of trans isomers of unsaturated monoene and diene FA, as well as C18:2n-6 in colostrum. Serum concentrations of IgG tended to be increased in calves born from dams fed fat compared with those not fed fat, and prepartum feeding of SAT tended to improve circulating concentrations of IgG in newborn calves above the feeding of ESS. Apparent efficiency of absorption of IgG was improved in calves born from dams fed fat, and SAT supplementation appeared more effective than supplementation with ESS. Feeding SAT prepartum may be of greater benefit based upon greater circulating IgG concentrations of calves after colostrum feeding. Feeding moderate amounts of saturated or unsaturated long-chain FA during the last 8 wk of gestation changed the FA profile of colostrum and plasma of neonates to reflect that of the supplements.

Effect of supplementing essential fatty acids to pregnant nonlactating Holstein cows and their preweaned calves on calf performance, immune response, and health.

The objective was to evaluate the effect of supplementing saturated or unsaturated fatty acids (FA) during late gestation of cows and during the preweaning period of calves on growth, health, and immune responses of calves. During the last 8wk of pregnancy, Holstein cattle (n=96) were fed no fat supplement (control), a saturated FA (SFA) supplement enriched in C18:0, or an unsaturated FA supplement enriched in the essential FA linoleic acid. Newborn calves were fed a milk replacer (MR) with either low linoleic acid (LLA; coconut oil) or high linoleic acid (HLA; coconut oil and porcine lard) concentration as the sole feedstuff during the first 30d. A grain mix with minimal linoleic acid was offered between 31 and 60d of life. At 30 and 60d of life, concentrations of linoleic acid in plasma were increased in calves born from dams supplemented with essential FA compared with SFA (44.0 vs. 42.5% of total FA) and in calves consuming HLA compared with LLA MR (46.3 vs. 40.8% of total FA). Total n-3 FA concentration was increased in plasma of calves fed HLA compared with LLA MR (1.44 vs. 1.32%) primarily due to increased α-linolenic acid. Prepartum supplementation with SFA tended to improve dry matter intake (48.8 vs. 46.7kg) and improved average daily gain (0.50 vs. 0.46kg/d) by calves without affecting efficiency of gain or circulating concentrations of anabolic metabolites or hormones. Increasing mean intake of linoleic acid from approximately 4.6 to 11.0g/d during the first 60d of life increased average daily gain (0.50 vs. 0.45kg/d) without a change in dry matter intake, thus improving feed efficiency (0.63 vs. 0.59kg of gain/kg of dry matter intake). Improved weight gain in calves fed HLA MR was accompanied by increased or tendency to increase plasma concentrations of glucose (92.7 vs. 89.9g/dL) and insulin-like growth factor I (59.5 vs. 53.2g/dL), increased hematocrit (36.0 vs. 34.4%) and concentration of blood lymphocytes (4.61 vs. 4.21×10(3)/µL), lowered plasma concentrations of acid-soluble protein (78.8 vs. 91.3mg/L) and blood platelets (736 vs. 822×10(3)/µL), and increased production of IFN-γ by peripheral blood mononuclear cells at 30d of age (48.1 vs. 25.6pg/mL), possibly indicating an earlier development of the immune system. Partial replacement of coconut oil with porcine lard in MR improved calf performance and some aspects of immunity.

Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows.

The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa(2+)) <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 43 g 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 iCa(2+) 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 72 h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa(2+) <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, ß-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa(2+) 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 iCa(2+) concentrations throughout the 24-h treatment period (0.77 ± 0.01 vs. 1.26 ± 0.01 mM iCa(2+)). Induction of SCH reduced dry matter intake on the day of infusion (5.3 ± 0.8 vs. 9.1 ± 0.8 kg/d) and rumen contractions (1.9 ± 0.2 vs. 2.7 ± 0.2 contractions/2 min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44 ± 0.23 vs. 2.32 ± 0.23 ng/mL) evident between 6 and 18 h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40 ± 0.04 vs. 4.17 ± 0.04 mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110 ± 0.019 vs. 0.061 ± 0.014 mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa(2+) 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.

Hormonal manipulations in the 5-day timed artificial insemination protocol to optimize estrous cycle synchrony and fertility in dairy heifers.

Objectives were to determine the effects of GnRH at the initiation of the 5-d timed artificial insemination (AI) program combined with 2 injections of PGF2α on ovarian responses and pregnancy per AI (P/AI) in dairy heifers, and the role of progesterone concentrations on LH release and ovulation in response to GnRH. In study 1, heifers received a controlled internal drug release (CIDR) insert containing 1.38 g of progesterone on d 0, an injection of 25 mg of PGF2α and CIDR removal on d 5, and an injection of 100 µg GnRH concurrently with AI on d 8. Heifers were assigned to receive no additional treatment (control; n=559) or an injection of GnRH on d 0 and a second injection of PGF2α on d 6 (G2P; n=547). In study 2, all heifers were treated as described for the control in study 1, and were allocated to receive no additional treatment (control; n=723), an injection of PGF2α on d 6 (NG2P; n=703), or an injection of GnRH on d 0 and an injection of PGF2α on d 6 (G2P; n=718). In study 3, heifers received a CIDR on d 7 after ovulation and were assigned randomly to a low-progesterone (LP; n=6) treatment in which 2 injections of 25 mg of PGF2α each were administered 12h apart, on d 7 and 7.5 after ovulation, or to a high-progesterone (HP; n=12) treatment in which no PGF2α was administered. On d 8, heifers received 100 µg of GnRH and blood was sampled at every 15 min from -30 to 180 min relative to the GnRH for assessment of LH concentrations. Additionally, 94 heifers were assigned to LP or HP and ovulation in response to GnRH was evaluated. In study 1, P/AI was greater for G2P than for the control on d 32 (59.4 vs. 53.5%) and 60 after AI (56.6 vs. 51.3%). In study 2, administration of GnRH on d 0 increased the proportion of heifers with a new corpus luteum on d 5 (control=21.9 vs. NG2P=20.1 vs. G2P=34.4%). Administration of a second PGF2α increased the proportion of heifers with progesterone <0.5 ng/mL at AI (control=83.1 vs. NG2P=93.0 and G2P=87.2%). Pregnancy per AI was greater for G2P than for control and NG2P on d 32 (control=52.9 vs. NG2P=55.0 vs. G2P=61.7%) and 60 (control=49.0 vs. NG2P=51.6 vs. G2P=59.1%). In study 3, HP attenuated LH release and reduced ovulation (19.0 vs. 48.4%) in response to GnRH compared with LP. Combining GnRH and 2 doses of PGF2α in the 5-d timed AI protocol improved follicle turnover, luteolysis, and P/AI in heifers. Elevated concentrations of progesterone suppressed LH release and are linked with the low ovulatory response to the initial GnRH treatment of the protocol.

Effect of feeding according to energy balance on performance, nutrient excretion, and feeding behavior of early lactation dairy cows.

The objectives of this study were to evaluate 2 feeding strategies for early lactation cows on performance and efficiency of nutrient utilization. Fifty-eight Holsteins cows were blocked by parity and production during the pretreatment period and then randomly assigned at 21 d postpartum to a control diet [n=29; 16.2% crude protein, 1.64 Mcal of net energy for lactation (NE(L)), 22% starch, and 19% forage neutral detergent fiber (NDF)] or a diet with caloric density manipulated weekly (precision diet; n=29; 16.2% crude protein; 1.59 to 1.68 NE(L); 18 to 26% starch; and 16 to 22% forage NDF) to promote a calculated positive energy balance of 5 Mcal/day. Diets were fed as total mixed rations and precision cows had their diets adjusted individually once a week, by feeding additional grain supplementation from 0 to 25% of daily dry matter (DM) offered, according to the energy balance of the preceding week. Energy balance was calculated daily and then averaged weekly. The study lasted from wk 3 to 19 postpartum, and nutrient digestibility, rumen fluid composition, urinary output, estimates of microbial protein synthesis, and feeding behavior were evaluated between wk 9 and 13 postpartum. Compared with controls, precision cows had similar DM intake (24.3 kg/d), but NE(L) intake tended to be greater primarily between wk 4 and 8 postpartum. Yields of milk (45.2 vs. 41.9 kg/d), milk components, 3.5% fat-corrected milk (44.0 vs. 40.8 kg/d), and energy-corrected milk (43.4 vs. 40.2) were all greater for precision than control cows, resulting in greater energy-corrected milk production per kilogram of diet DM consumed (1.79 vs. 1.72). Precision cows produced more milk calories per kilogram of metabolic weight (0.227 vs. 0.213 Mcal of NE(L)/kg), although the amount of consumed calories partitioned into milk (82.3%) and measures of energy status did not differ between treatments throughout the study. Glucose concentrations were greater throughout the day in precision cows compared with controls at 6 wk, but not 13 wk postpartum. Apparent digestibility of nutrients, composition of rumen fluid, mean and low rumen pH, and estimated rumen microbial N synthesis remained mostly unaltered by treatments. Although precision cows produced more milk true protein, measures of efficiency of dietary N use were not influenced by treatment. On wk 13 postpartum, precision cows consumed a diet with longer NDF particles, which resulted in a tendency for greater intake of NDF >8mm because of less sorting against the long particles than control cows. Meal pattern differed with treatment, and precision cows consumed feed more sparsely throughout the day, spent more time ruminating lying, and had similar meal duration (mean of 36.3 min/meal) compared with control cows, but smaller meal size (3.33 vs. 3.64 kg/meal). Results from the current study indicate that allocating dietary resources according to the individual needs of cows based on energy balance improves lactation performance compared with feeding a single total mixed ration, despite similar average nutrient intake between treatments. Improvements in performance are likely related to allocation of calories based on the needs of the cow and on shifts of feeding behavior that might favor intake of smaller meals.

Targeted progesterone supplementation improves fertility in lactating dairy cows without a corpus luteum at the initiation of the timed artificial insemination protocol.

The objectives of this study were to determine the effect of supplemental progesterone on fertility in lactating dairy cows lacking a corpus luteum (CL) at the initiation of the timed artificial insemination (AI) program. Holstein cows were subjected to the 5-d timed AI program (d -8 GnRH, d -3 and -2 PGF2α, d 0 GnRH and AI). Cows had their ovaries scanned by ultrasonography on d -8 and those bearing a CL were considered to be in diestrus (DI; n=946). Cows that lacked a CL on d -8 were assigned to remain as untreated control (CON; n=234) or receive 2 controlled internal drug release (CIDR) inserts containing progesterone (2CIDR; n=218) from d -8 to -3, as a single insert has been proven insufficient to modulate fertility in cows without CL. Blood was analyzed for progesterone and estradiol concentrations. Pregnancy was diagnosed on d 34 and 62 after AI. Progesterone concentrations during the timed AI program were lowest for CON, intermediate for 2CIDR, and highest for DI. Supplementation increased progesterone concentrations between d -7 and -3 compared with CON (2.65 vs. 0.51 ng/mL). Ovulation to the first GnRH was not affected by treatment. However, a greater proportion of CON and 2CIDR cows had a new CL on d -3 compared with DI cows (66.7 vs. 61.9 vs. 52.0%). In cows with a new CL, the diameter of the ovulatory follicle was larger for CON than 2CIDR, and intermediate for DI (18.7 vs. 16.5 vs. 17.7 mm). Concentrations of estradiol on d -3 did not differ among treatments; however, DI cows had greater estradiol concentrations at AI compared with CON or 2CIDR cows. Pregnancy per AI was less for CON compared with 2CIDR or DI on d 32 (30.8 vs. 46.8 vs. 49.9%) and 64 (28.6 vs. 43.7 vs. 47.3%), indicating that supplementation with progesterone reestablished fertility in cows lacking a CL on d -8. A greater proportion of nonpregnant CON cows had a short reinsemination interval compared with 2CIDR or DI (11.1 vs. 3.5 vs. 5.7%). Treatment did not affect pregnancy loss between d 34 and 62 of gestation. A single ultrasound exam was effective in identifying a low-fertility cohort of cows based on the absence of CL at the first GnRH injection of the timed AI protocol. Progesterone supplementation with 2 CIDR inserts increased progesterone in plasma to 2.65 ng/mL and restored fertility in lactating dairy cows lacking a CL at the initiation of the timed AI program similar to that of cows in diestrus.

Prevalence of periparturient diseases and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates.

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 30 d 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 ß-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70 mM, subclinical ketosis if the ß-hydroxybutyrate concentration was ≥0.96 mM, and subclinical hypocalcemia if the Ca concentration was ≤2.14 mM. 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, 86 d 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.

Effects of 1 or 2 treatments with prostaglandin F2α on subclinical endometritis and fertility in lactating dairy cows inseminated by timed artificial insemination.

The objectives of the current study were to investigate the efficacy of PGF2α as a therapy to reduce the prevalence of subclinical endometritis and improve pregnancy per artificial insemination (P/AI) in cows subjected to a timed artificial insemination (AI) program. A total of 1,342 lactating Holstein dairy cows were allocated randomly at 25 ± 3 d in milk (DIM) to remain as untreated controls (control, n=454) or to receive a single PGF2α treatment at 39 ± 3 DIM (1PGF, n=474) or 2 treatments with PGF(α at 25 ± 3 and 39 ± 3 DIM (2PGF, n=414). All cows were enrolled in the double Ovsynch program at 48 ± 3 DIM and were inseminated at 75 ± 3 DIM. A subset of 357 cows had uterine samples collected for cytological examination at 25 ± 3, 32 ± 3, and 46 ± 3 DIM to determine the percentage of polymorphonuclear leukocytes (PMNL). Subclinical endometritis was defined by the presence of ≥ 5% PMNL. Vaginal discharge score was evaluated at 25 ± 3 DIM and used to define the prevalence of purulent vaginal discharge. Body condition score was assessed at 25 ± 3 DIM. Pregnancy was diagnosed 32 d after AI and reconfirmed 28 d later. At 32 ± 3 DIM, the prevalence of subclinical endometritis was reduced by treatment with PGF2α at 25 ± 3 DIM in 2PGF (control=23.5% vs. 1PGF=28.3% vs. 2PGF=16.7%); however, this benefit disappeared at 46 ± 3 DIM, and 14% of the cows remained with subclinical endometritis. One or 2 treatments with PGF2α did not influence P/AI on d 32 or 60 after timed AI, which averaged 39.9 and 35.2%. Similarly, treatment with PGF2α had no effect on pregnancy loss between 32 and 60 d of gestation (11.9%). Cows diagnosed with both purulent vaginal discharge and subclinical endometritis had the lowest P/AI and the highest pregnancy loss compared with those diagnosed with only 1 of the 2 diseases or compared with cows having no diagnosis of uterine diseases. Interestingly, subclinical endometritis depressed P/AI and increased pregnancy loss only when it persisted until 46 DIM. On d 32 after AI, cows not diagnosed with subclinical endometritis and those that resolved subclinical endometritis by 46 DIM had greater P/AI than those that remained with subclinical endometritis at 46 DIM (45.4 and 40.0 vs. 25.0%, respectively). Similar to P/AI, cows not diagnosed with subclinical endometritis and those that resolved subclinical endometritis by 46 DIM had less pregnancy loss than those with subclinical endometritis at 46 DIM (9.6 and 13.5 vs. 43.9%, respectively). One or 2 treatments with PGF2α before initiation of the timed AI program were unable to improve uterine health, P/AI, and maintenance of pregnancy in lactating dairy cows. Cows diagnosed with both purulent vaginal discharge and subclinical endometritis had the greatest depressions in measures of fertility at first AI, particularly when subclinical endometritis persisted in the early postpartum period.