E.S. Ribeiro

Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease

In this prospective cohort study, Holstein cows considered to be at high risk of developing metritis (dystocia, twins, stillbirth, retained placenta, or their combination) were matched with herdmates at low risk of developing metritis (normal calving) and monitored daily for rectal temperature and uterine discharge during the first 12 d in milk (DIM). Blood was sampled on d 0, 1, and 3 postpartum for assessment of neutrophil phagocytic and oxidative burst activities. Blood was also sampled at 0, 1, 2, 3, 4, 7, and 12 DIM for determination of serum concentrations of Ca, K, Mg, nonesterified fatty acids, β-hydroxybutyrate, and glucose. On the basis of receiver operator characteristic curves, subclinical hypocalcemia (SCH) was defined as a serum Ca concentration ≤8.59 mg/dL in at least 1 sample in the first 3 DIM. The overall incidences of metritis and puerperal metritis were 47.3 and 30%, respectively. Concentration in blood and percentages of neutrophils undergoing phagocytosis and oxidative burst were all reduced in cows with SCH compared with normocalcemic cows. Cows with SCH were at a greater risk of developing fever, metritis, and puerperal metritis compared with normocalcemic cows. Among cows at low risk of developing metritis, those with SCH had a greater incidence of metritis (40.7%) compared with normocalcemic cows (14.3%). Similarly, among cows at high risk of developing metritis, cows with SCH had a greater incidence of metritis (77.8%) compared with normocalcemic cows (20.0%). Cows with SCH had elevated concentrations of nonesterified fatty acids and β-hydroxybutyrate compared with normocalcemic cows. The relative risk of developing metritis decreased by 22% for every 1mg/dL increase in serum Ca. Neither metritis nor SCH influenced the resumption of estrous cyclicity by 38 DIM, but cows with SCH had a reduced pregnancy rate and a longer interval to pregnancy compared with normocalcemic cows. Finally, the population risk to develop uterine diseases attributable to SCH was 66.6% for metritis and 91.3% for puerperal metritis in the present study.

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 β-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70 mM, subclinical ketosis if the β-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.

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, β-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic 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.

Effect of interval between induction of ovulation and artificial insemination (AI) and supplemental progesterone for resynchronization on fertility of dairy cows subjected to a 5-d timed AI program

Objectives were to investigate 2 intervals from induction of ovulation to artificial insemination (AI) and the effect of supplemental progesterone for resynchronization on fertility of lactating dairy cows subjected to a 5-d timed AI program. In experiment 1, 1,227 Holstein cows had their estrous cycles presynchronized with 2 injections of PGF(2α) at 46 and 60 d in milk (DIM). The timed AI protocols were initiated with GnRH at 72 DIM, followed by 2 injections of PGF(2α) at 77 and 78 DIM and a second injection of GnRH at either 56 (OVS56) or 72h (COS72) after the first PGF(2α) of the timed AI protocols. All cows were time-inseminated at 72h after the first PGF(2α) injection. Pregnancy was diagnosed on d 32 and 60 after AI. In experiment 2, 675 nonpregnant Holstein cows had their estrous cycles resynchronized starting at 34 d after the first AI. Cows received the OVS56 with (RCIDR) or without (RCON) supplemental progesterone, as an intravaginal insert, from the first GnRH to the first PGF(2α). Pregnancy diagnoses were performed on d 32 and 60 after AI. During experiment 2, subsets of cows had their ovaries scanned by ultrasonography at the first GnRH, the first PGF(2α), and second GnRH injections of the protocol. Blood was sampled on the day of AI and 7 d later, and concentrations of progesterone were determined in plasma. Cows were considered to have a synchronized ovulation if they had progesterone <1 and >2.26 ng/mL on the day of AI and 7 d later, respectively, and if no ovulation was detected between the first PGF(2α) and second GnRH injections during resynchronization. In experiment 1, the proportion of cows detected in estrus at AI was greater for COS72 than OVS56 (40.6 vs. 32.4%). Pregnancy per AI (P/AI) did not differ between OVS56 (46.4%) and COS72 (45.5%). In experiment 2, cows supplemented with progesterone had greater P/AI compared with unsupplemented cows (51.3 vs. 43.1%). Premature ovulation tended to be greater for RCON than RCIDR cows (7.5 vs. 3.6%), although synchronization of the estrous cycle after timed AI was similar between treatments. Timing of induction of ovulation with GnRH relative to insemination did not affect P/AI of dairy cows enrolled in a 5-d timed AI program. Furthermore, during resynchronization starting on d 34 after the first AI, supplementation with progesterone improved P/AI in cows subjected to the 5-d timed AI protocol.

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.

Effects of presynchronization and length of proestrus on fertility of grazing dairy cows subjected to a 5-day timed artificial insemination protocol.

The objectives were to compare the effects of 2 methods of presynchronization and 2 lengths of proestrus on fertility of grazing dairy cows subjected to a 5-d timed artificial insemination (AI) protocol at initiation of breeding season. Lactating dairy cows (n=1,754) from 3 seasonal grazing farms were blocked within farm by breed, parity, and days in milk (DIM). Study d 0 was considered the day of AI of cows in COS72 (72h of proestrus). Within each block, cows were randomly assigned to 1 of 2 presynchronization treatments: a PGF(2α)-based program, Presynch, consisting of 2 injections of PGF(2α) administered on d -32 and -18, or a PGF(2α)-GnRH-based program, Double-Ovsynch (DO), consisting of GnRH on d -25, PGF(2α) on d -18, and GnRH on d -15. Within each of the 2 presynchronization treatments, cows were randomly assigned to 1 of 2 lengths of proestrus within the 5-d timed AI protocol, consisting of GnRH on d -8, PGF(2α) on d -3 and -2, and GnRH+AI at either 58 h (COS58) or 72 h (COS72) after the d -3 PGF(2α) injection. Ovaries were scanned by ultrasonography twice, on d -42 and -32, to determine estrous cyclicity before enrollment in the study. Blood was sampled and analyzed for concentrations of estradiol on the day of AI. Pregnancies per AI (P/AI) were determined 30 and 65 d after AI. Presynchronization did not affect the concentration of estradiol at AI (DO=6.4 vs. Presynch=5.8 pg/mL), detection of estrus at AI (20.8 vs. 25.9%), or P/AI on d 30 (56.8 vs. 59.1%) and 65 (52.5 vs. 52.4%) after the first AI. Cows receiving COS72 had increased concentration of estradiol (6.6 vs. 5.5 pg/mL) and detection of estrus at AI (28.5 vs. 10.8%) compared with cows receiving COS58. Length of proestrus did not affect P/AI on d 30 (COS72=58.7 vs. COS58=56.1%) but, in Presynch cows, COS58 was detrimental to fertility on d 65 after AI (54.9 vs. 46.5%). Pregnancy loss between gestational d 30 and 65 was greater for Presynch than for DO (7.6 vs. 11.3%), but it was not affected by length of proestrus. Estrous cyclic cows had greater P/AI than anovular cows on d 30 (61.7 vs. 35.1%) and 65 (56.1 vs. 30.7%), but no interaction between estrous cyclic status and treatments was detected. Crossbred Holstein/Jersey cows had superior fertility than their purebred counterparts during the breeding season. The Presynch and DO protocols resulted in similar fertility with no overall difference between the presynchronization methods; however, limiting the length of proestrus to 58 h reduced P/AI in the 5-d timed AI protocol when cows had their estrous cycle presynchronized with Presynch but not with DO.

Fertility in dairy cows following presynchronization and administering twice the luteolytic dose of prostaglandin F2α as one or two injections in the 5-day timed artificial insemination protocol

The objectives were to evaluate pregnancy per AI (P/AI) of dairy cows subjected to the 5-day timed AI protocol under various synchronization and luteolytic treatments. Cows were either presynchronized or received supplemental progesterone during the synchronization protocol, and received a double luteolytic dose of PGF2α, either as one or two injections. In Experiment 1, dairy cows (n=737; Holstein=250, Jersey=80, and crossbred=407) in two seasonal grazing dairy farms were randomly assigned to one of four treatments in a 2×2 factorial arrangement. The day of AI was considered study Day 0. Half of the cows were presynchronized (G6G: PGF2α on Day -16 and GnRH on Day -14) and received the 5-day timed AI protocol using 1 mg of cloprostenol, either as a single injection (G6G-S: GnRH on Day -8, PGF2α on Day -3, and GnRH+AI on Day 0) or divided into two injections of 0.5 mg each (G6G-T: GnRH on Day -8, PGF2α on Day -3 and -2, and GnRH+AI on Day 0). The remaining cows were not presynchronized and received a controlled internal drug-release (CIDR) insert containing progesterone from GnRH to the first PGF2α injection of the 5-day timed AI protocol, and 1 mg of cloprostenol either as a single injection on Day -3 (CIDR-S) or divided into two injections of 0.5 mg each on Days -3 and -2 (CIDR-T). Ovaries were examined by ultrasonography on Days -8 and -3 and plasma progesterone concentrations were determined on Days -3 and 0. In Experiment 2, 655 high-producing Holstein cows had their estrous cycle presynchronized with PGF2α at 46±3 and 60±3 days postpartum and were randomly assigned to receive 50 mg of dinoprost during the 5-day timed AI protocol, either as a single injection or divided into two injections of 25 mg each. Pregnancies per AI were determined on Days 35 and 64 after AI in both experiments. In Experiment 1, presynchronization with G6G increased the proportion of cows with a CL on Day -8 (80.6 vs. 58.8%), ovulation to the first GnRH of the protocol (64.2 vs. 50.2%), and the presence (95.6 vs. 88.4%) and number (1.79 vs. 1.30) of CL at PGF(2α) compared with CIDR cows. Luteolysis was greater for two injections compared to a single PGF2α injection (two PGF2α=95.9 vs. single PGF2α=72.2%), especially in presynchronized cows (G6G-T=96.2 vs. G6G-S=61.7%). For cows not presynchronized, two PGF2α injections had no effect on P/AI (CIDR-S=30.2 vs. CIDR-T=34.3%), whereas for presynchronized cows, it improved P/AI (G6G-S=28.7 vs. G6G-T=45.4%). In Experiment 2, the two-PGF2α injection increased P/AI on Days 35 (two PGF2α=44.5 vs. single PGF2α=36.4%) and 64 (two PGF2α=40.3% vs. single PGF2α=32.6%) after AI. Presynchronization and dividing the dose of PGF2α (either cloprostenol or dinoprost) into two injections increased P/AI in lactating dairy cows subjected to the 5-day timed AI protocol.

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.

Low Doses of Bovine Somatotropin Enhance Conceptus Development and Fertility in Lactating Dairy Cows

ABSTRACT Objectives were to evaluate the effects of administering either one or two low doses of slow-release recombinant bovine somatotropin (bST) on hormone concentrations, conceptus development, and fertility in dairy cows. Cows from two farms were detected in estrus on or after 50 days postpartum (n = 1483), inseminated, and enrolled in the study (Day 0). Within farm, cows were blocked by parity and assigned randomly to receive a single placebo injection at insemination (control), a single injection with 325 mg of bST at insemination (S-bST), or two injections with 325 mg of bST administered on Days 0 and 14 (T-bST). From a subset of cows, blood was collected twice weekly from Day 0 to 42 for determination of hormone concentrations and on Day 19 for isolation of leucocytes and analysis of transcript abundance of selected interferon-stimulated genes. Pregnancy was diagnosed on Days 31 and 66, and ultrasonographic morphometry of the conceptus was performed on Days 34 and 48 in a subset of cows. Cows that received T-bST had increased plasma concentrations of GH and IGF1 for 4 wk, increased mRNA expression of ISG15 and RTP4 in leukocytes, earlier rise in the pregnancy-specific protein B in plasma of pregnant cows, increased conceptus size, and enhanced fertility. Cows that received S-bST had increased concentrations of GH and IGF1 for only 2 wk and it was insufficient to alter conceptus development and fertility. In conclusion, supplementation with low doses of bST during the pre- and peri-implantation periods enhanced conceptus development, reduced embryonic losses, and improved fertility in dairy cows.

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AI (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AI on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF(2α) and removal of the CIDR on d 5, and GnRH concurrent with timed AI on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF(2α) injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF(2α) did not differ between treatments and averaged 87.1%. Progesterone on the day of AI was greater for GnRH than control (0.50 ± 0.07 vs. 0.28 ± 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GnRH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF(2α) and removal of the CIDR on d 5, and either GnRH 56 h after PGF(2α) and AI 16h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF(2α) (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but CO S72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF(2α) injection 5 d later. Moreover, extending the proestrus by delaying the final GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.