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

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

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

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

Effects of maternal choline supplementation on performance and immunity of progeny from birth to weaning.

The objectives were to investigate whether supplementation with rumen-protected choline (RPC) during late pregnancy in Holstein cows affects offspring immunity and growth, and whether effects are utero-placental, colostrum dependent, or both. A total of 105 multiparous Holstein cows were assigned randomly to a prepartum diet (1.54 Mcal of NEL/kg of DM, and 15.8% CP) without (control) or with added RPC (12.9 g/d of choline ion). Calves (n = 111) were blocked by sex and assigned randomly to colostrum from control cows or colostrum from RPC cows, resulting in 4 treatments in a 2 × 2 factorial arrangement: (1) calves born and fed colostrum from non-supplemented dams (NN; n = 33); (2) calves from non-supplemented dams and fed colostrum from RPC-fed cows (NC; n = 25); (3) calves from RPC-supplemented dams and colostrum from non-supplemented cows (CN; n = 28); and (4) calves from RPC-supplemented dams and colostrum from RPC-fed cows (CC; n = 25). Growth, intakes, and immunity of females were evaluated up to 56 d of age. Growth and intake of male calves was evaluated up to 35 d of age, and physiological and immune responses to intravenous LPS challenge were evaluated from 21 to 35 d of age. Effects of prenatal and colostrum treatments and interactions between treatments were analyzed using mixed models. Calves fed colostrum from RPC-supplemented dams had a 17.4% increase in apparent efficiency of absorption of IgG compared with calves fed colostrum from control dams (27.4 vs. 23.3%). Incidence of fever in the first 21 d of age tended to be less in females born from RPC-supplemented dams compared with females born from control dams (31 vs. 58%). Prenatal RPC females had increased hematocrit and concentrations of red blood cells, leukocytes, neutrophils, and lymphocytes in blood compared with prenatal females born from control dams. Compared with prenatal control females, prenatal RPC females had greater intake of milk replacer (704 vs. 748 ± 9.9 g/d) and starter (45.4 vs. 60.2 ± 5.9 g/d) during the first 21 d of age. In male calves, mean intake of DM was greater (1,074 vs. 976 ± 45 g/d) after the LPS challenge (0 to 8 d) by calves born from dams fed RPC compared with males born from control dams. Calves born from RPC-fed dams also had lower mean rectal temperature (39.0 vs. 39.2°C) and mean respiration rate (35.6 vs. 39.3 breaths/min) compared with males born from control dams. Moreover, serum concentrations of metabolites (i.e., ß-hydroxybutyric acid, fatty acids, and glucose), cytokines (i.e., tumor necrosis factor-α) and acute phase proteins (i.e., serum amyloid A) were consistent with less-severe inflammatory response to LPS in males born from dams fed RPC compared with control. Source of colostrum and interaction between prenatal and colostrum treatments had minimal effects on calf responses to LPS. Overall, maternal RPC supplementation during late gestation suggests a positive effect on immunity, in that colostrum from RPC-fed dams increased efficiency of IgG absorption and maternal supplementation with RPC during late gestation, regardless of colostrum source, attenuated responses to LPS.

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.

Effects of timing of artificial insemination and treatment of semen with a Slo3 potassium channel blocker on fertility of dairy heifers subjected to the 5-day CIDR-Synch protocol.

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.

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.

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 ß-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.

Hepatic mRNA expression for genes related to somatotropic axis, glucose and lipid metabolisms, and inflammatory response of periparturient dairy cows treated with recombinant bovine somatotropin.

Objectives of this experiment were to evaluate the effects of recombinant bovine somatotropin (rbST) treatment of periparturient dairy cows on hepatic mRNA expression for genes related to the somatotropic axis, insulin, glucose, and lipid metabolism, inflammation, and oxidative stress. Holstein cows were enrolled in the experiment at 253 ± 3 d of gestation and assigned to 1 of 3 treatments: untreated control (n = 53), 87.5 mg of rbST (n = 56; rbST87.5), and 125 mg of rbST (n = 57; rbST125). Cows in the rbST87.5 and rbST125 treatments received weekly injections of rbST from -21 to 28 d relative to calving. A subsample of cows (control = 20, rbST87.5 = 20, rbST125 = 20) was randomly selected for collection of liver samples according to expected calving date, BCS, and previous lactation 305-d mature equivalent milk yield. Only cows that had liver sampled at -21 ± 3, -7 ± 3, and 7 ± 3 d relative to calving were used in the current experiment. Blood, sampled weekly from -28 to 21 d relative to calving, was used to determine the concentrations of growth hormone, insulin-like growth factor 1, insulin, cortisol, fatty acids, ß-hydroxybutyrate, glucose, haptoglobin, and tumor necrosis factor-α. Liver samples were used to determine hepatic mRNA expression of 50 genes. Treatment with rbST increased growth hormone concentrations during the postpartum period (control = 9.0 ± 0.7, rbST87.5 = 15.3 ± 1.0, rbST125 = 18.5 ± 1.3 ng/mL) and increased insulin-like growth factor 1 concentrations during the prepartum period (control = 107.4 ± 7.2, rbST87.5 = 126.9 ± 6.6, rbST125 = 139.4 ± 6.9 ng/mL). Control cows had greater postpartum concentrations of ß-hydroxybutyrate (control = 776.4 ± 64.0, rbST87.5 = 628.4 ± 59.7, rbST125 = 595.4 ± 60.9 µmol/L) than rbST cows. The rbST87.5 and rbST125 treatments upregulated the hepatic mRNA expression for somatotropic axis genes (GHR, GHR1A, IGF1, IGFBP3, and SOCS2) on d -7 relative to calving and upregulated the mRNA expression for SOCS2 on d 7. On d -7, rbST87.5 and rbST125 treatments increased mRNA expression for genes involved in hepatic lipid transport (ANGPTL4, APOA5, APOB100, and SCARB1) and downregulated mRNA expression for PPARD, which is involved in lipid storage. On d 7, rbST tended to upregulate the mRNA expression for genes involved in gluconeogenesis (PCK1) and fatty acid ß-oxidation (ACOX1), and downregulated the mRNA expression for genes involved in inflammation (TNFRSF1A, ICAM1, CXCL1, MYD88, HIF1A, IL1RN, NFKBIA, and SOCS3) and oxidative stress (XBP1). Administration of rbST during the periparturient period may improve liver function and health by increasing hepatic capacity for gluconeogenesis and lipid transport and by reducing inflammation and oxidative stress.

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.

Synchronized ovulation for first insemination improves reproductive performance and reduces cost per pregnancy in dairy heifers.

The objectives were to evaluate the effects of synchronizing estrus and ovulation to implement a timed artificial insemination (AI) at first insemination on reproductive performance and cost per pregnancy in dairy heifers. Six hundred eleven Holsteins heifers at approximately 400 d of age from 3 farms were enrolled in the study. Six days before moving to the breeding pens, heifers were allocated randomly to AI after detected estrus from study d 0 to 84 (CON, n=306), or to timed AI for first AI followed by detected estrus for the remainder of the 84-d study (TAI, n=305). Heifers receiving TAI were enrolled in the 5-d timed AI protocol on study d -6 (d -6, GnRH and a progesterone insert; d -1, PGF2α and insert removal; d 0, PGF2α; d 2, GnRH + AI), and they were allowed to be bred the day before scheduled timed AI if detected in estrus. Starting on study d 0, estrus was detected daily. Heifers in estrus were inseminated on the same morning as detected estrus. Control heifers not inseminated by study d 7 received PGF2α and this treatment was repeated every 2 wk until AI. The study lasted 84 d to allow a period of breeding equivalent to four 21-d estrous cycles. A herd budget accounting for inputs for both treatments was created to determine the cost per pregnancy. Sensitivity analysis compared economic differences between the 2 treatments under different input scenarios when detection of estrus after the first AI varied from 50 to 80%. Interval to first AI was 8 d shorter for TAI than for CON. Pregnancy at first AI did not differ between treatments (CON=58.3 vs. TAI=62.8%). In contrast, TAI increased pregnancy per AI (P/AI) compared with CON in heifers inseminated with sex-sorted semen (CON=31.6 vs. TAI=54.8%). The 21-d cycle insemination rate was greater for TAI (91.4%) than for CON (82.4%), even when evaluated after the first 21 d in the study (CON=68.2 vs. TAI=77.1%). The increased insemination rate improved the 21-d cycle pregnancy rate from 47.9% in CON to 57.2% in TAI heifers. In fact, the hazard of pregnancy was 60% greater for TAI than CON. The increased pregnancy rate in TAI reduced the median days to pregnancy by 12 d (CON=2.0 vs. TAI=14.0) and increased the proportion of pregnant heifers by 6.3 percentage points by study d 84 (CON=85.2 vs. TAI=91.5%). The cost per pregnancy was $17.00 less for TAI than CON. The sensitivity analysis indicated that TAI was economically more advantageous to produce a pregnancy compared with CON. Only when insemination rate after the first 21 d of breeding was very high and P/AI was relatively low did the cost per pregnancy become similar for the 2 treatments. Collectively, inseminating all heifers within 2 d of breeding with the 5-d timed AI protocol maintains P/AI, improves pregnancy rate, and reduces cost per pregnancy compared with insemination after detected estrus.

Effects of intrauterine infusion of Trueperella pyogenes on endometrial mRNA expression of proinflammatory cytokines and luteolytic cascade genes and their association with luteal life span in dairy cows.

Objectives were to determine the effects of intrauterine infusion (IUI) of Trueperella pyogenes on endometrial expression of proinflammatory cytokines and luteal life span. Holstein cows (n = 32) were allocated randomly, in two replicates (15 then 17 cows), to receive one of three treatments on Day 5 of the estrous cycle: TP (n = 13), IUI containing 10(9) colony-forming units/mL of T. pyogenes; tumor necrosis factor (TNF; n = 9), IUI containing 1 µg of TNFα; and control (n = 10), IUI of saline solution. Five cows per treatment had uterine biopsies collected at 6, 12, and 24 hours after treatment to evaluate the endometrial messenger RNA expression of TNFα (TNF), interleukin-1ß (IL1B), IL6, IL8, prostaglandin E synthase (PGES), prostaglandin F synthase (PGFS), and oxytocin receptor (OXR), and histologic evidence of inflammation. Messenger RNA expression was measured using quantitative reverse transcription polymerase chain reaction. The remaining cows had ovaries scanned and blood collected for progesterone evaluation; however, only seven, four, and three cows in the TP, TNF, and control groups were used for comparison in replicate 2. The GLIMMIX procedure of SAS was used for statistical analysis. All TP and TNF cows had moderate to severe endometrial inflammation, whereas only one control had mild inflammation. Premature luteolysis occurred in three, one, and zero cows in the TP, TNF and control groups, respectively. Delayed luteolysis occurred in one TP and one TNF cow. Interleukin-1ß expression was greater in the TP cows than in the TNF cows at 24 hours after IUI. Moreover, IL6 expression tended to be greater for the TP cows than for the control cows at 12 hours after IUI. Interleukin 8 expression was greater in the TP cows than in the control and TNF cows at 24 hours after IUI. Oxytocin receptor expression tended to be greater for the TP cows and was greater for the TNF cows than for the control cows at 12 hours. The messenger RNA expressions of TNF, PGES, and PGFS were not affected by treatment, time, or their interaction. In conclusion, IUI of T. pyogenes or TNFα led to histologic evidence of inflammation and early luteolysis in some cows, which may have been caused by increased endometrial expression of proinflammatory cytokines (i.e., IL1B, IL6), chemokines (i.e., IL8), and luteolytic cascade factors (i.e., OXR).

Meta-analysis of progesterone supplementation during timed artificial insemination programs in dairy cows.

A systematic review of the literature was performed with the objective to evaluate the effects of progesterone supplementation using a single intravaginal insert during timed artificial insemination (AI) programs on fertility in lactating dairy cows. A total of 25 randomized controlled studies including 8,285 supplemented cows and 8,398 untreated controls were included in the meta-analysis. Information regarding the presence of corpus luteum (CL) at the initiation of the synchronization protocol was available for 6,883 supplemented cows and 6,879 untreated controls in 21 experiments. Studies were classified based on service number (first AI vs. resynchronized AI), use of presynchronization (yes vs. no), and insemination of cows in estrus during the synchronization protocol (inseminated in estrus and timed AI vs. timed AI only). Reproductive outcomes of interest were pregnancy per AI (P/AI) measured on d 32 (27 to 42) and 60 (41 to 71) after AI, and pregnancy loss between d 32 and 60 of gestation. Random effects meta-analyses were conducted and treatment effect was summarized into a pooled risk ratio with the Knapp-Hartung modification (RRK+H). The effect of moderator variables was assessed using meta-regression analyses. Progesterone supplementation increased the risk of pregnancy on d 32 [RRK+H = 1.08; 95% confidence interval (CI) = 1.02-1.14] and 60 after AI (RRK+H = 1.10; 95% CI = 1.03-1.17). The benefit of progesterone supplementation was observed mainly in cows lacking a CL at the initiation of the timed AI program (d 60: RRK+H = 1.18; 95% CI = 1.07-1.30) rather than those with CL (d 60: RRK+H = 1.06; 95% CI = 0.99-1.12). Progesterone supplementation benefited P/AI in studies in which all cows were inseminated at timed AI (d 60: RRK+H = 1.20; 95% CI = 1.10-1.29), but not in studies in which cows could be inseminated in estrus during the timed AI program (d 60: RRK+H = 1.04; 95% CI = 0.92-1.16). Progesterone supplementation tended to reduce the risk of pregnancy loss (RRK+H = 0.84; 95% CI = 0.67-1.00). Service number and presynchronization did not influence the effect of progesterone supplementation on fertility. In summary, progesterone supplementation using a single intravaginal insert during the timed AI program increased P/AI mostly in cows without CL and reduced the risk of pregnancy loss in lactating dairy cows. Insemination of cows in estrus during the synchronization protocol eliminated the benefit of supplemental progesterone on P/AI.

Progesterone supplementation to lactating dairy cows without a corpus luteum at initiation of the Ovsynch protocol.

The objectives were to determine the effect of progesterone supplementation on fertility responses in lactating dairy cows without corpora lutea (CL) at initiation of the timed artificial insemination (AI) program. Holstein cows from 5 commercial dairy farms were subjected to the Ovsynch-56 protocol (d -10 GnRH, d -3 PGF2α, d -0.7 GnRH, d 0 AI). Ovaries were scanned by ultrasonography on d -10. Within farm, cows without CL were blocked by pen and assigned randomly to remain as nonsupplemented controls (CON; n = 652) or to receive 2 controlled internal drug-release (CIDR) inserts containing 1.38 g of progesterone each from d -10 to -3 (2CIDR; n = 642). Cows with CL were randomly selected within pen and used as positive controls as cows in diestrus at the initiation of the Ovsynch protocol (DIEST; n = 640). Signs of estrus were detected beginning on d -9 based on removal of tail chalk, and cows in estrus received AI on the same day. Blood samples from subsets of cows on d -10, -9, -7, -5, -3, and 0 (n = 109) and on d 6, 13, and 19 (n = 156) were analyzed for progesterone concentrations. Pregnancy was diagnosed on d 32 and 60 after AI. The average progesterone concentration during the timed AI program was lowest for CON, intermediate for 2CIDR, and highest for DIEST (0.92, 2.77, and 4.93 ng/mL, respectively). The proportions of cows that ovulated in response to the first GnRH (63.6, 61.1, and 47.2%, respectively) and that had a new CL on d -3 at PGF2α injection (72.4, 67.9, and 47.4%, respectively) were greater for CON and 2CIDR compared with DIEST, respectively. The diameter of the ovulatory follicle and the proportion of cows that ovulated in response to the second GnRH did not differ among treatments. A greater proportion of CON and 2CIDR cows were detected in estrus at AI compared with DIEST cows (35.8, 39.6, and 30.6%, respectively). Pregnancy per AI was less for CON compared with 2CIDR and DIEST on d 32 (31.3, 42.2, and 38.4%, respectively) and d 60 after AI (28.9, 37.2, and 33.9%, respectively), indicating that progesterone supplementation reestablished fertility in cows lacking a CL similar to that of cows in diestrus at the initiation of the timed AI program. Treatment did not affect pregnancy loss between d 32 and 60 of gestation. Pregnancy from a subset of cows with plasma progesterone concentrations indicated that a minimum concentration of 2.0 ng/mL was needed to optimize fertility. A single ultrasound examination effectively identified a low-fertility cohort of cows based on the absence of CL at the first GnRH injection of the Ovsynch protocol. Supplementation with 2 CIDR inserts increased progesterone in plasma by an additional 1.85 ng/mL compared with CON, resulting in concentrations of 2.77 ng/mL during development of the ovulatory follicle, which restored fertility in dairy cows lacking CL to a level similar to that of cows in diestrus.

Effect of progesterone supplementation on fertility responses of lactating dairy cows with corpus luteum at the initiation of the Ovsynch protocol.

The objectives of this study were to determine the effects of supplemental progesterone on fertility responses of lactating dairy cows with a CL at the initiation of the timed artificial insemination (AI) program. Holstein cows were subjected to the Ovsynch protocol (Day -10 GnRH, Day -3 PGF2α, Day -0.7 GnRH, and Day 0 timed AI). Ovaries were scanned by ultrasonography on Day -10 and cows with CL were blocked by pen and assigned randomly to receive no supplemental progesterone (control, n = 863) or to receive a controlled internal drug-release (CIDR) insert containing progesterone from Days -10 to -3 (1CIDR, n = 862). Cows were observed for signs of estrus beginning on Day -9 based on removal of tail chalk and those in estrus received AI on the same day. Blood sampled from a subset of cows was analyzed for progesterone concentrations on Days -10, -9, -7, -5, -3, 0, 6, 13, and 19. Pregnancy was diagnosed on Days 32 and 60 after AI. Supplementation increased (P < 0.01) progesterone concentrations between Day -9 and -3 compared with control (7.5 vs. 6.2 ng/mL). Treatment had no effect on the ovulatory response to the first and final GnRH injections of the Ovsynch protocol, the proportion of cows that maintained their CL until the day of PGF2α injection, or the diameter of the ovulatory follicle before AI. Although the overall proportion of cows in estrus at AI did not differ between treatments, progesterone supplementation prevented (P < 0.01) cows from coming into estrus from Days -9 to -3 (0.0% vs. 4.7%), whereas it increased (P = 0.02) the proportion of cows inseminated in estrus from Days -2 to -1 (7.1% vs. 4.5%). Pregnancy per AI (P/AI) tended to be reduced (P = 0.06) by supplemental progesterone on Day 32 (40.5% vs. 45.0%), but not on Day 60 after AI (36.6% vs. 39.7%). A tendency for an interaction (P = 0.09) between treatment and the presence of CL at the PGF2α injection was observed for P/AI on Day 32 in cows that received timed AI because progesterone supplementation reduced P/AI in cows that maintained their CL until Day -3 (40.3% vs. 46.7%); however, it increased P/AI in those that did not have a CL at PGF2α (38.1% vs. 27.7%). Treatment did not affect pregnancy loss between Days 32 and 60 of gestation. In conclusion, incorporating a single intravaginal insert to the timed AI program increased progesterone concentrations in plasma by 1.3 ng/mL, but did not benefit fertility in dairy cows that have CL at the initiation of the synchronization protocol.

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.

Effects of supplemental progesterone after artificial insemination on expression of interferon-stimulated genes and fertility in dairy cows.

The objectives of the current study were to evaluate the effects of supplemental progesterone after artificial insemination (AI) on expression of IFN-stimulated genes (ISG) in blood leukocytes and fertility in lactating dairy cows. Weekly cohorts of Holstein cows were blocked by parity (575 primiparous and 923 multiparous) and method of insemination (timed AI or AI on estrus) and allocated randomly within each block to untreated controls, a controlled internal drug release (CIDR) containing 1.38g of progesterone from d 4 to 18 after AI (CIDR4), or a CIDR on d 4 and another on d 7 after AI and both removed on d 18 (CIDR4+7). Blood was sampled to quantify progesterone concentrations in plasma and mRNA expression in leukocytes for the ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) and receptor transporter protein-4 (RTP4) genes. Pregnancy was diagnosed on d 34±3 and 62±3 after AI. Treatment increased progesterone concentrations between d 5 and 18 after AI in a dose-dependent manner (control=3.42, CIDR4=4.97, and CIDR4+7=5.46ng/mL). Cows supplemented with progesterone tended to have increased luteolysis by d 19 after AI (control=17.2; CIDR4=29.1; CIDR4+7=30.2%), which resulted in a shorter AI interval for those reinseminated after study d 18. Pregnancy upregulated expression of ISG in leukocytes on d 19 of gestation, but supplementing progesterone did not increase mRNA abundance for ISG15 and RTP4 on d 16 after insemination and tended to reduce mRNA expression on d 19 after AI. For RTP4 on d 19, the negative effect of supplemental progesterone was observed only in the nonpregnant cows. No overall effect of treatment was observed on pregnancy per AI on d 62 after insemination and averaged 28.6, 32.7, and 29.5% for control, CIDR4, and CIDR4+7, respectively. Interestingly, an interaction between level of supplemental progesterone and method of AI was observed for pregnancy per AI. For cows receiving exogenous progesterone, the lower supplementation with CIDR4 increased pregnancy per AI on d 62 in cows inseminated following timed AI (CIDR4=39.2; CIDR4+7=27.5%); in those inseminated following detection of estrus, however, the use of a second insert on d 7 resulted in greater pregnancy per AI (CIDR4=26.9; CIDR4+7=31.5%). Pregnancy loss did not differ among treatments. Supplemental progesterone post-AI using a single intravaginal insert on d 4 was beneficial to pregnancy in cows inseminated following timed AI, but incremental progesterone with a second insert on d 7 did not improve fertility of dairy cows.

Inducing ovulation early postpartum influences uterine health and fertility in dairy cows.

The objective of the current study was to evaluate the effect of GnRH early postpartum on induction of ovulation, uterine health, and fertility in dairy cows. Holstein cows without a corpus luteum (CL) at 17 ± 3 DIM were assigned randomly to receive i.m. GnRH (n = 245) at 17 ± 3 and 20 ± 3 DIM or remain as controls (n = 245). Ovaries were scanned by ultrasonography twice weekly totaling 4 examinations. Ovulation was characterized by the appearance of a CL ≥ 20 mm at any ultrasound or CL <20mm in 2 consecutive examinations. Clinical and cytological endometritis were diagnosed at 35 DIM. Compared with control, GnRH increased ovulation up to 3.5d after the last treatment (78.7 vs. 45.0%) and did not affect the prevalence of clinical endometritis (23.9 vs. 18.6%) or cytological endometritis (30.9 vs. 32.8%). Prevalence of clinical endometritis increased in cows that had calving problems (32.6 vs. 15.9%) and metritis (40.6 vs. 15.8%). Metritis increased prevalence of cytological endometritis (50.7 vs. 23.5%). Treatment with GnRH did not affect pregnancy per artificial insemination at 32 (37.6 vs. 38.6%) or 74 d after artificial insemination (35.0 vs. 31.5%), but reduced pregnancy loss (6.8 vs. 18.1%). No overall effect of GnRH treatment on hazard of pregnancy was observed; however, an interaction between GnRH treatment and ovulation showed that GnRH-treated cows that ovulated had increased hazard of pregnancy by 300 DIM compared with GnRH-treated and control cows that did not ovulate (hazard ratio=2.0 and 1.3, respectively), but similar to control cows that ovulated (hazard ratio=1.1). Gonadotropin-releasing hormone early postpartum induced ovulation without affecting uterine health, but failed to improve pregnancy per artificial insemination or time to pregnancy, although it reduced pregnancy loss.

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.

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.