Effects of synchronization treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid hormone concentrations in lactating dairy cows

Lactating dairy cows (n=57) ≥45 d postpartum at first service were enrolled in a randomized complete block design study to evaluate treatments to synchronize estrus and ovulation. At 10 d before artificial insemination (AI), animals were randomly assigned to 1 of 3 treatments: (1) d -10 GnRH (GnRH1; 10 μg of buserelin, i.m.) and controlled internal drug release insert [CIDR, 1.38 g of progesterone (P4)]; d -3 PGF(2α) (PGF; 25 mg of dinoprost, i.m.); d -2 CIDR out; and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH (GnRH2) 36 h after CIDR out and timed AI (TAI) 18 h later (CIDR_TAI); or (3) same as CIDR_TAI, but no CIDR (Ovsynch). Transrectal ultrasound was used to assess follicle size before ovulation and on d 4, 8, and 15 after the presumptive day of estrus (d 0) to measure the corpus luteum (CL). Blood samples were collected to determine concentrations of estradiol (E2; d -10, -9, -3, -2, -1, and 0) and P4 (d -10, -9, -2, -1, 0, 1, 4, 6, 8, 11, and 15). No treatment differences were observed in either circulating concentrations of P4 or the ovulatory response to GnRH1 at the onset of synchronization treatments. Circulating concentrations of P4 were greater for CIDR_OBS and CIDR_TAI compared with Ovsynch at 24 h after CIDR insertion (5.34 and 4.98 vs. 1.75 ng/mL) and immediately before CIDR removal (1.65 and 1.48 vs. 0.40 ng/mL). Peak circulating concentrations of E2 were greater for CIDR_OBS compared with Ovsynch (3.85 vs. 2.39 pg/mL), but CIDR_TAI (2.82 pg/mL) did not differ from either CIDR_OBS or Ovsynch. The interval from PGF injection to peak circulating E2 did not differ between CIDR_TAI and Ovsynch (52.1 vs. 49.8 h). Both CIDR_TAI and Ovsynch, however, had shorter intervals from PGF injection to peak circulating E2 concentrations compared with CIDR_OBS (67.8 h). The diameter of the dominant follicle before ovulation was greater for CIDR_OBS compared with Ovsynch (18.5 vs. 16.0 mm) but CIDR_TAI (17.1 mm) did not differ from either of the other treatments. The mean interval from PGF to ovulation was longer for CIDR_OBS (100.0 h) compared with CIDR_TAI and Ovsynch (84.4 and 83.2 h, respectively). Use of CIDR_OBS resulted in increased preovulatory follicle size and greater circulating concentrations of E2 due to a longer period of preovulatory follicle growth. Progesterone supplementation during synchronization and GnRH on the day before TAI affected ovulatory follicle size, and periovulatory circulating concentrations of P4 and E2. No differences, however, in postovulatory P4 or luteal volume profiles were observed.     

Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows

Ovulation to the first GnRH injection of Ovsynch-type protocols is lower in cows with high progesterone (P4) concentrations compared with cows with low P4 concentrations, suggesting that P4 may suppress the release of LH from the anterior pituitary after GnRH treatment. The objectives of this study were to determine the effect of 1) circulating P4 concentrations at the time of GnRH treatment on GnRH-induced LH secretion in lactating dairy cows and 2) increasing the dose of GnRH from 100 to 200 μg on LH secretion in a high- and low-P4 environment. A Double-Ovsynch (Pre-Ovsynch: GnRH, PGF(2α) 7d later, GnRH 3d later, and Breeding-Ovsynch 7d later: GnRH, PGF(2α) 7d later, and GnRH 48 h later) synchronization protocol was used to create the high- and low-P4 environments. At the first GnRH injection of Breeding-Ovsynch (high P4), all cows with a corpus luteum ≥ 20 mm were randomly assigned to receive 100 or 200 μg of GnRH. At the second GnRH injection of Breeding-Ovsynch (low P4) cows were again randomized to receive 100 or 200 μg of GnRH. Blood samples were collected every 15 min from -15 to 180 min after GnRH treatment, and then hourly until 6h after GnRH treatment. As expected, mean P4 concentrations were greater for cows in the high- than the low-P4 environment. For cows receiving 100 μg of GnRH, the LH peak and area under the curve (AUC) were greater in the low- than in the high-P4 environment. Similarly, for cows receiving 200 μg of GnRH, the LH peak and AUC were greater in the low- than the high-P4 environment. Cows receiving 100 or 200 μg of GnRH had greater mean LH concentration in the low- than the high-P4 environment from 1 to 6h after GnRH treatment. On the other hand, when comparing the effect of the 2 GnRH doses in the high- and low-P4 environments, cows receiving 200 μg of GnRH had a greater LH peak and AUC than cows treated with 100 μg of GnRH both in the high- and low-P4 environments. For the high-P4 environment, mean LH was greater from 1.5 to 5h after GnRH treatment for cows receiving 200 μg of GnRH than for those receiving 100 μg of GnRH. In the low-P4 environment, mean LH was greater for cows receiving 200 μg of GnRH than for those receiving 100 μg of GnRH from 1 to 2.5h after GnRH treatment. We conclude that the P4 environment at GnRH treatment dramatically affects GnRH-induced LH secretion, and that a 200-μg dose of GnRH can increase LH secretion in either a high- or a low-P4 environment.     

Characterization of the variability and repeatability of gonadotropin-releasing hormone–induced luteinizing hormone responses in dairy cows within a synchronized ovulation protocol

The primary objective was to determine the variability and repeatability of GnRH-induced LH responses. The secondary objective was to evaluate the associations among plasma LH, FSH, estradiol (E2), and progesterone (P4) concentrations. One hundred lactating Holstein cows (35 primiparous, 65 multiparous) were initially subjected to a presynchronization protocol (d 0, PGF_2α; d 3, GnRH) followed 7 d later by Ovsynch (d 10, GnRH; d 17, PGF_2α; 56 h later, GnRH) and timed artificial insemination 16 h after the last GnRH. Blood samples were collected immediately before the GnRH injection of presynchronization and the second GnRH of Ovsynch to determine plasma concentrations of LH, FSH, and P4. A second blood sample was collected 2 h after each of the above GnRH injections to determine GnRH-induced LH and FSH concentrations. Plasma concentrations of E2 were also determined in samples collected immediately before the second GnRH of Ovsynch. Cows that (1) had higher LH concentrations at 0 h than at 2 h after GnRH, (2) showed an ongoing spontaneous LH surge, (3) did not respond to GnRH, and (4) had P4 ≥ 0.5 ng/mL at GnRH of presynchronization and the second GnRH of Ovsynch were excluded from the analysis. The variability (coefficient of variation) and repeatability [between animal variance/(within animal variance + between animal variance)] of GnRH-induced LH response were determined from samples collected 2 h after the GnRH of presynchronization and the second GnRH of Ovsynch. The associations among plasma LH, FSH, E2, and P4 were determined at the second GnRH of Ovsynch. Mean (±SEM) LH concentrations before GnRH were 0.5 ± 0.04 and 0.6 ± 0.03 ng/mL, whereas mean LH concentrations 2 h after GnRH were 9.8 ± 1.0 and 12.1 ± 0.8 ng/mL at GnRH of presynchronization and the second GnRH of Ovsynch, respectively. The variability of GnRH-induced LH was 76.1 and 52.1% at GnRH of presynchronization and the second GnRH of Ovsynch, respectively. The repeatability estimate for GnRH-induced LH concentration between GnRH of presynchronization and Ovsynch assessments was 0.10. Plasma concentrations of LH were positively associated with FSH and E2 (r = 0.61 and 0.30, respectively) and negatively associated with P4 (r = ?0.46) at the second GnRH of Ovsynch. In summary, GnRH-induced LH responses were highly variable and unrepeatable, and LH concentrations were positively associated with FSH and E2 and negatively associated with P4.     

Interventions after artificial insemination: conception rates, pregnancy survival, and ovarian responses to gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone

We hypothesized that increasing concentrations of progesterone (P4) after artificial insemination would increase fertility. Our objective was to assess changes in ovarian structures, incidence of ovulation, and change in serum P4 in response to GnRH, human chorionic gonadotropin (hCG), or exogenous P4 (controlled internal drug release; CIDR insert) treatment beginning 4 to 9 d after artificial insemination (d 0) and again 7 d later (experiment 1). Blood was collected from 753 cows in 3 herds on d 0 and 7. Ovaries of 162 cows were scanned and mapped to confirm the presence of a corpus luteum (CL), and cows were assigned randomly to serve as controls (n = 41) or to receive a CIDR insert for 7 d (n = 41), 100 μg of GnRH (n = 40), or 3,300 IU of hCG (n = 40). More cows were induced to ovulate in response to GnRH (60%) and hCG (78%) compared with controls (2.4%). Compared with controls, cows treated with GnRH or hCG had more induced CL (d 7) and more total CL (d 7), but serum P4 was increased only in response to hCG. Largest follicle diameters on d 7 were less after GnRH and hCG, but total follicular volume on d 7 was reduced by GnRH, hCG, and CIDR, compared with that of controls. Volume of the original luteal structures was increased by hCG but tended to be reduced by CIDR and GnRH compared with luteal volume in controls. Total CL volume was increased by hCG, but reduced by CIDR, compared with CL volume of controls. Conception rates and pregnancy survival were assessed in response to the same treatments described in experiment 1: controls (n = 708), CIDR (n = 711), GnRH (n = 719), and hCG (n = 714). Tendencies for interactions of treatment × herd and treatment × lactation group were detected, but no 3-way interactions were found. Treatment with hCG increased conception rates in second-lactation cows. The CIDR tended to increase, and hCG increased, conception rates in 2 herds, whereas the CIDR decreased conception rates in 1 herd. Pregnancy survival was reduced by GnRH compared with that in controls. We concluded that GnRH and hCG effectively induced ovulation, and increased number of CL, but only increased serum P4 in hCG-treated cows. Further, treatment with the CIDR or hCG increased conception rates but only in some herds.     

Resynchronization of ovulation protocols for dairy cows including or not including gonadotropin-releasing hormone to induce a new follicular wave: Effects on re-insemination pattern,ovarian responses,and pregnancy outcomes

Our objectives were to evaluate the pattern of re-insemination, ovarian responses, and pregnancy per artificial insemination (P/AI) of cows submitted to different resynchronization of ovulation protocols. The base protocol started at 25 ± 3 d after artificial insemination (AI) and was as follows: GnRH, 7 and 8 d later PGF_2α, GnRH 32 h after second PGF_2α, and fixed timed AI (TAI) 16 to 18 h after GnRH. At 18 ± 3 d after AI, cows were randomly assigned to the G25 (n = 1,100) or NoG25 (n = 1,098) treatments. The protocol for G25 and NoG25 was the same, except that cows in NoG25 did not receive GnRH 25 ± 3 d after AI. At nonpregnancy diagnosis (NPD), 32 ± 3 d after AI, cows from G25 and NoG25 with a corpus luteum (CL) ≥15 mm in diameter and a follicle ≥10 mm completed the protocol (G25 CL = 272, NoG25 CL = 194), whereas cows from both treatments that did not meet these criteria received a modified Ovsynch protocol with P4 supplementation [controlled internal drug release insert plus GnRH, controlled internal drug release insert removal, and PGF_2α 7 and 8 d later, GnRH 32 h after second PGF_2α, and TAI 16 to 18 h after GnRH (G25 NoCL = 53, NoG25 NoCL = 78)]. Serum concentrations of progesterone (P4) were determined and ovarian ultrasonography was performed thrice weekly from 18 ± 3 d after AI until 1 d after TAI (G25 = 46, NoG25 = 44 cows). A greater percentage of NoG25 cows were re-inseminated at detected estrus (NoG25 = 53.5%, G25 = 44.6%), whereas more cows had a CL at NPD in G25 than NoG25 (83.7 and 71.3%). At 32 d after AI, P/AI was similar for G25 and NoG25 for inseminations at detected estrus (38.4 and 42.9%), TAI services for cows with no CL (40.4 and 36.7%), and for all services combined (39.6 and 39.0%). However, P/AI were greater for cows with a CL in G25 than NoG25 (40.6 and 32.8%) that received TAI. More cows ovulated spontaneously or in response to GnRH for the G25 than the NoG25 treatment (70 and 36%) but a similar proportion had an active follicle at NPD (G25 = 91% and NoG25 = 96%). The largest follicle diameter at NPD (G25 = 15.0 ± 0.4 mm, NoG25 = 16.5 ± 0.6 mm) and days since it reached ≥10 mm (G25 = 4.0 ± 0.3 d, NoG25 = 5.8 ± 0.6 d) were greater for the NoG25 than G25 treatment. For cows with a CL at NPD, CL regression after NPD, ovulation after TAI, and ovulatory follicle diameter did not differ. In conclusion, removing the first GnRH of a modified Resynch-25 protocol for cows with a CL at NPD and a modified Ovsynch protocol with P4 supplementation for cows without a CL at NPD resulted in a greater percentage of cows re-inseminated at detected estrus and a similar proportion of cows pregnant in spite of reduced P/AI for cows with a CL at NPD.     

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d ?11, PGF_2α on d ?4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF_2α) on d ?2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d ?11, 1 CIDR withdrawn + PGF_2α on d ?4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF_2α on d ?2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d ?11 or d ?4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF_2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF_2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF_2α treatment (d ?4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF_2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.     

A GnRH/LH surge without subsequent progesterone exposure can induce development of follicular cysts

Our hypothesis was that follicular cysts would develop if cows experienced an estradiol-induced GnRH LH surge in the absence of an ovulatory follicle. Further, we hypothesized that estradiol would fail to induce a subsequent GnRH/LH surge in these cows until they were treated with progesterone. In experiment 1, seven cows were synchronized with a controlled internal drug releasing device (CIDR) for 9 d and each received 500 microg of cloprostenol on d 7. All follicles (> or = 5 mm in diameter) were aspirated at the time of CIDR removal using transvaginal follicular aspiration. Two days after aspiration, cows were treated with 5 mg of estradiol benzoate (EB) to induce a GnRH/LH surge in the absence of an ovulatory-sized follicle. All cows had an LH surge following the estradiol treatment and three of seven developed an anovulatory condition that resembled follicular cysts. The four cows that did not develop follicular cysts luteinized remaining cells from one aspirated follicle each. Thus, all cows with a progesterone elevation after the estradiol/GnRH/LH surge had subsequent ovulatory cycles, whereas the absence of progesterone was followed by follicular cysts. After 49 d, the anovulatory cows were induced back to normal cyclicity by insertion of a CIDR for 7 d. In two subsequent experiments, nine of 26 cows were induced to have follicular cysts by follicular aspiration followed by 5 mg of EB. After 26 d of observation, all cystic cows received a second treatment with 5 mg of EB and none of the cows showed an LH surge or ovulation. Cystic cows were untreated (n = 4 controls) or treated for 7 d with a CIDR (n = 5). All cystic cows were subsequently treated for a third time with 5 mg of EB. All CIDR-treated cows had an LH surge and ovulated, whereas none of the control cows had an LH surge or ovulation after the estradiol treatment. Thus, a large follicle anovulatory condition, similar to follicular cysts, can be induced by estradiol induction of a GnRH/LH surge in the absence of subsequent luteinization, and this condition prevents a GnRH/LH surge in response to high doses of estradiol. Progesterone eliminates this condition by reinitiation of GnRH/LH surges in response to estradiol.     

Level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows

The objective of this experiment was to determine the effect of high versus low progesterone (P4) during the pre-dominance or dominance phase (or both) of ovulatory follicle development on follicular dynamics and fertility of lactating dairy cows. Progesterone (P4) was manipulated to reach high (H) or low (L) serum concentrations during the pre-dominance phase (d 0 to 4 of the wave) and dominance phase (d 5 to 7 of the wave) of a second follicular wave ovulatory follicle, creating 4 treatments: H/H, H/L, L/H, and L/L. Luteolysis was induced with PGF_2α on d 7 of the wave and ovulation was induced with GnRH 56 h after PGF_2α. Cows (n = 558) received artificial insemination (AI) 16 h following GnRH. Pregnancy was determined at 6 intervals during gestation and at calving to quantify pregnancy loss beginning at d 23 post-AI utilizing pregnancy-specific protein B (PSPB) in novel within-cow comparisons. Cows with single ovulations assigned to the L/L treatment had greater pre-ovulatory follicle diameter compared with cows assigned to the L/H or H/L treatments. Cows with single ovulations had greater pre-ovulatory follicle diameter compared with cows with double ovulations. Low P4 in H/L, L/H, and L/L increased double ovulation rate compared with H/H. Cows with double ovulations had greater pregnancies per AI (P/AI) on d 23 post-AI compared with cows with single ovulations but had greater losses if ovulations were unilateral. Cows with low P4 during the entire period of the ovulatory follicle development also had greater P/AI on d 23 post-AI compared with cows with high P4 during both phases. However, full-term P/AI was not different between treatments. This was a result of the greater incidence of pregnancy losses between d 35 and 56 of gestation for cows with unilateral double ovulations compared with bilateral double ovulations and single ovulatory cows. Cows with single ovulation and low circulating P4 during the dominance period of follicle development had increased pregnancy losses between d 35 and 56 of gestation compared with cows with single ovulations and high P4. The PSPB measurements on d 16 and 23 post-AI were highly accurate in the prediction of pregnancy at d 28. The PSPB differed on d 23 and 28 between cows that had versus cows that did not have pregnancy losses between d 28 and 35 of gestation. In summary, circulating concentrations of P4 during ovulatory follicle development affected numbers of follicles ovulated and timing of subsequent pregnancy losses.     

Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination

The objective was to determine if using a Double-Ovsynch protocol [DO; Pre-Resynch: GnRH-7 d-PGF(2α)-3 d-GnRH, 7 d later Breeding-Resynch: GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-timed artificial insemination (TAI)] to resynchronize ovulation after a previous TAI would increase synchrony and pregnancies per AI (P/AI) compared with an Ovsynch protocol initiated 32 d after TAI (D32; GnRH-7 d-PGF(2α)-56 h-GnRH-16 h-TAI). Lactating Holstein cows at various days in milk and prior AI services were blocked by parity and randomly assigned to resynchronization treatments. All DO cows received the first GnRH injection of Pre-Resynch 22 d after TAI, and cows (n=981) diagnosed not pregnant using transrectal ultrasonography 29 d after TAI continued the protocol. Pregnancy status for all D32 cows was evaluated 29 d after TAI so fertility and pregnancy loss could be compared with that of DO cows. All D32 cows received the first GnRH injection of Ovsynch 32 d after TAI, and cows (n=956) diagnosed not pregnant using transrectal palpation 39 d after TAI continued the protocol. In a subgroup of cows from each treatment, ultrasonography (n=751) and serum progesterone (P4) concentrations (n=743) were used to determine the presence of a functional corpus luteum (CL) and ovulation to the first GnRH injection of D32 and Breeding-Resynch of DO (GnRH1), luteal regression after PGF before TAI, and ovulation to the GnRH injection before TAI (GnRH2). Overall, P/AI 29 d after TAI was not affected by parity and was greater for DO compared with D32 cows (39 vs. 30%). Pregnancy loss from 29 to 74 d after TAI was not affected by parity or treatment. The percentage of cows with a functional CL (P4 ≥1.0 ng/mL) at GnRH1 was greater for DO than D32 cows (81 vs. 58%), with most DO cows having medium P4 (60%; 1.0 to 3.49 ng/ml), whereas most D32 cows had either low (42%; <1.0 ng/mL) or high (36%; ≥3.5 ng/mL) P4 at GnRH1. Ovulation to GnRH1 was similar between treatments but was affected by serum P4 at GnRH. Cows with low P4 (<1.0 ng/mL) had the greatest ovulatory response (59%), followed by cows with medium (≥1.0 to 3.49 ng/mL; 38%) and then high (≥3.50 ng/mL; 16%) P4 at GnRH1. A greater percentage of DO cows were synchronized compared with D32 cows (72 vs. 51%) primarily due to a greater percentage of D32 than DO cows without a functional CL at the PGF injection before TAI (35 vs. 17%) or without complete CL regression before GnRH2 (17 vs. 7%). We conclude that DO increased fertility of lactating dairy cows during a resynchronization program primarily by increasing synchronization of cows during the Ovsynch protocol before TAI.     

Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows

Two experiments evaluated the influence of follicular wave at artificial insemination (AI) on fertility of dairy cows. In experiment 1, data from 5,607 lactating cows enrolled in estrous and ovulation synchronization programs for AI were evaluated. Cows’ blood was analyzed for progesterone 7 to 14 d apart, with the second sample collected on the day of the first GnRH (GnRH1) of the synchronization protocol. Cows were classified as cyclic if progesterone was ≥1 ng/mL in at least 1 of the 2 samples and as anovular if both samples were <1 ng/mL. Cyclic cows were categorized as low (CLOW; < 1 ng/mL) or high (CHIGH; ≥ 1 ng/mL) progesterone on the day of GnRH1, which would result in ovulation of the dominant follicle of the first (FW) and second (SW) follicular waves, respectively, at AI. Pregnancy per AI (P/AI) was determined 30 and 53 d after AI. In experiment 2, 220 cyclic Holstein cows received 2 injections of PGF_2α administered 14 d apart. The Ovsynch protocol (d 0 GnRH, d 7 PGF_2α, d 9 GnRH, d 9.5 timed AI) was initiated either 3 or 10 d after the second PGF_2α of the presynchronization to result in insemination to the FW or SW dominant follicles. Blood was analyzed for progesterone and ovaries were scanned to determine ovulatory responses and follicle diameter. Pregnancy was determined on d 32 and 67 after timed AI. In experiment 1, P/AI on d 30 was greater for CHIGH cows than for CLOW and anovular cows (43.0, 31.3, and 29.7%, respectively), but because of pregnancy loss, P/AI on d 53 was lowest for anovular cows. Proportions of cows with short reinsemination intervals differed among groups and were 7.1, 15.7, and 11.9% for CHIGH, CLOW, and anovular cows, respectively. Pregnancy loss was greater for anovular cows than for CLOW cows (15.0 vs. 10.0%) and was intermediate for CHIGH cows (13.5%). In experiment 2, 9.8 and 97.2% of the FW and SW cows, respectively, had progesterone ≥1 ng/mL at GnRH1. Concentrations of progesterone at the GnRH1 and PGF_2α injections of the Ovsynch protocol were greater for SW cows than FW cows. Pregnancy per AI was greater for SW cows than for FW cows (41.7 vs. 30.4%) despite less ovulation to GnRH1 in SW cows than in FW cows (78.7 vs. 88.4%). Collectively, these data indicate that follicular wave of the ovulatory follicle and not cyclic status caused the greatest reduction in P/AI in dairy cows. Whether the culprit is the follicle itself or the hormonal milieu characteristic of the first follicular wave and the early stage of the estrous cycle remains to be elucidated. Synchronization programs that induced ovulation of the FW follicle at AI reduced P/AI in lactating dairy cows, and ovulation of the FW follicle, or development of the ovulatory follicle under low progesterone concentrations, or both, might be mechanisms for reduced fertility in anovular cows.     

Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.     

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation,follicle dynamics,and circulating follicle-stimulating hormone in lactating dairy cows

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 μg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (～18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5–11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.     

Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses

Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF_2α metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF_2α 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d −16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n = 19), cows had their estrous cycle synchronized starting on study d −9 (GnRH and progesterone insert on d −9, and insert removal and PGF_2α on d −2). In experiment 2 (n = 25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d −4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2 mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2, estradiol and progesterone concentrations did not differ between treatments from d 0 to 16; however, the proportion of cows with a short luteal phase tended to increase in LP than HP (25 vs. 0%). Concentrations of PGFM were greater for LP than HP. Uterine biopsies had a greater abundance of progesterone receptor, and tended to have less estrogen receptor-α abundance on d 12 compared with d 16. An interaction between treatment and day of collection was detected for estrogen receptor-α because of an earlier increase in protein abundance on d 12. Reduced concentrations of progesterone during the development of the ovulatory follicle altered follicular dynamics and follicular fluid composition, increased basal LH concentrations, and prematurely increased estrogen receptor-α abundance and exacerbated PGF_2α release in the subsequent estrous cycle.     

The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch,compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF_2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF_2α–14 d–PGF_2α–10 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. Treated cows received PGF_2α and GnRH–7 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. All cows received a supplemental injection of PGF_2α 24 h after the PGF_2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF_2α of Ovsynch. The administration of PGF_2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF_2α of Ovsynch, total luteal area (mm²), or serum concentrations of P4 at time of PGF_2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF_2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.     

Ovarian follicular responses to high doses of pulsatile luteinizing hormone in lactating dairy cattle

Two experiments in lactating dairy cows examined ovarian follicular responses to high, frequent doses of exogenous LH pulses at levels associated with follicular cysts. In Experiment 1, estrus was synchronized in 12 cyclic lactating cows >40 d postpartum. Emergence of the second follicular wave (d 0) was determined by ultrasonography. Starting on d 1, cows received LH (40 microg/h; n = 7) or saline (2 mL/h; n = 5) in hourly pulses for up to 5 (n = 5) or 7 (n = 7) d. On d 2, all cows received two injections of PGF2alpha, 12 h apart. In experiment 2, 14 lactating cows (7 to 12 d postpartum) received LH (40 microg/h; n = 7) or saline (1 mL/h; n = 7) in hourly pulses for 7 d, beginning 24 h after start of the first follicular wave. Daily samples were used to determine serum concentrations of progesterone (P4), estradiol-17beta (E2), LH, and FSH. Profiles of LH were determined from blood samples collected at 12-min intervals for 8 h on d 3. During infusion of LH, serum P4 and FSH were similar across treatments in both experiments. Serum E2 concentrations were similar in experiment 1, but serum E2 was greater on d 2, 3, and 5 in LH-treated cows in experiment 2. Infusion increased LH pulse frequency and amplitude in both experiments. Formation of cysts did not differ between LH- and saline-treated cows in either experiment (1 of 7 vs. 0 of 5 and 1 of 6 vs. 0 of 7, respectively). Cows that ovulated had similar intervals to ovulation in experiment 1 [6.0 +/- 0.1 d (LH) vs. 6.4 +/- 0.2 d (saline)], but in experiment 2, ovulation was 14 d earlier in LH-treated cows (5.6 +/- 1.8 d vs 19.9 +/- 1.5 d). In conclusion, high concentrations of LH are not solely responsible for formation of cysts in lactating dairy cows. Pulsatile infusion of LH stimulated follicular growth and steroidogenesis and decreased time to first ovulation in anestrous postpartum cows.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Effects of additional gonadotropin-releasing hormone and prostaglandin F2α treatment to an estradiol/progesterone-based embryo transfer protocol for recipient lactating dairy cows

This study was designed to evaluate whether the utilization of a second PGF_2α treatment at the end of an estradiol/progesterone (E2/P4)-based protocol with or without GnRH at the beginning of the protocol would improve pregnancy rates of lactating Holstein cows assigned to timed embryo transfer. A total of 501 lactating Holstein cows in 5 farms were enrolled in the experiment. Within farm, cows were blocked by parity and, within block, were assigned randomly to (1) insertion of an intravaginal P4 device (controlled internal drug-releasing device; CIDR) and estradiol benzoate on d ?11, PGF_2α on d ?4, CIDR withdrawal and an injection of estradiol cypionate on d ?2, and timed embryo transfer on d 7 (1-PGF; n = 164); (2) the same treatments as 1-PGF, but with PGF_2α administered on d ?4 and ?2 (2-PGF; n = 171); and (3) 2-PGF with the addition of a GnRH treatment on d ?11 (GnRH⁺2-PGF; n = 166). Ovaries were scanned by transrectal ultrasonography on d ?11, ?4, and 7, and blood samples were collected on d ?11, ?4, 0, and 7 for P4 determination. Treatment comparisons were performed using contrasts. The proportion of cows with a new corpus luteum on d ?4 was greater in GnRH⁺2-PGF cows. Cows in 1-PGF had a greater P4 concentration on d 0 but lesser P4 on d 7 compared with cows in the other groups. Cows assigned to receive 2-PGF (2-PGF and GnRH⁺2-PGF) had greater estrus expression, and a greater proportion of cows ovulated to estradiol cypionate. No further contrast effects were observed for follicle diameter, double ovulation rate, pregnancy per embryo transfer (P/ET) on d 32 and 60, or pregnancy loss. As P4 concentration on d ?4 increased, P/ET on d 60 tended to increase. Cows with P4 ≥3.66 ng/mL on d ?4 had greater P/ET on d 32 and 60 than those with P4 below that threshold. Regardless of treatment, cows with P4 concentration ≥3.66 ng/mL also had a greater pregnancy per synchronized protocol (P/SP) on d 60. Also, a P4 concentration on d ?4 (low or high) × follicle diameter (continuous) interaction tendency was observed when evaluating P/ET. Although P/ET did not differ among cows with different follicles sizes with reduced P4 concentration on d ?4 (<3.66 ng/mL), it increased in cows with larger follicles exposed to increased P4 concentration (≥3.66 ng/mL). When P4 on d 0 was evaluated, P/ET on d 32 and 60 was greater for cows with low (≤0.09 ng/mL) versus high (>0.21 ng/mL) P4; as P4 concentration on d 0 increased, P/ET linearly decreased. In summary, cows with increased P4 concentrations during growth of the ovulatory follicular wave had improved P/ET. Administering a second PGF_2α dose reduced P4 concentration on d 0 and increased ovulatory response to the protocol, but no benefits were observed on P/ET or P/SP.     

Effect of human chorionic gonadotrophin administration 2 days after insemination on progesterone concentration and pregnancy per artificial insemination in lactating dairy cows

The aim of this study was to examine the effect of a single administration of human chorionic gonadotrophin (hCG) during the establishment of the corpus luteum (CL) on progesterone (P4) concentration and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Postpartum spring-calving lactating dairy cows (n = 800; mean ± SD days in milk and parity were 78.5 ± 16.7 and 2.3 ± 0.8, respectively) on 3 farms were enrolled on the study. All cows underwent the same fixed-time AI (FTAI) protocol involving a 7-d progesterone-releasing intravaginal device with gonadotrophin-releasing hormone (GnRH) administration at device insertion, prostaglandin at device removal followed by GnRH 56 h later, and AI 16 h after the second GnRH injection. Cows were blocked on days postpartum, body condition score, and parity and randomly assigned to receive either 3,000 IU of hCG 2 d after FTAI or no further treatment (control). Blood samples were collected on d 7 and 14 postestrus by coccygeal venipuncture on a subset of 204 cows to measure serum P4 concentration, and pregnancy was diagnosed by ultrasonography approximately 30 and 70 d after FTAI. Administration of hCG caused an increase in circulating P4 concentrations compared with the control treatment on d 7 (+22.2%) and d 14 (+25.7%). The P/AI at 30 d after FTAI was affected by treatment, farm, body condition score, and calving to service interval. Overall, administration of hCG decreased P/AI (46.3% vs. 55.1% for the control). Among cows that did not become pregnant following AI, a greater proportion of control cows exhibited a short repeat interval (≤17 d) compared with cows treated with hCG (8.6% vs. 2.8%, respectively). In addition, the percentages of cows pregnant at d 21 (59.6% vs. 52.0%) and d 42 (78.3% vs. 71.9%) were greater in control than in hCG-treated cows. The overall incidence of embryo loss was 10.7% and was not affected by treatment. There was a tendency for an interaction between treatment and CL status at synchronization protocol initiation for both P4 concentration and P/AI. In conclusion, administration of hCG 2 d after FTAI increased circulating P4 concentrations. Unexpectedly, cows treated with hCG had lower fertility; however, this negative effect on fertility was manifested primarily in cows lacking a CL at the onset of the synchronization protocol.     

Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows

Haemodynamic changes are involved in the cyclic remodelling of ovarian tissue that occurs during final follicular growth, ovulation and new corpus luteum development. The aim of this study was to characterize the real-time changes in the blood flow within the follicle wall associated with the LH surge, ovulation and corpus luteum development in cows. Normally cyclic cows with a spontaneous ovulation (n = 5) or a GnRH-induced ovulation (n = 5) were examined by transrectal colour and pulsed Doppler ultrasonography to determine the area and the time-averaged maximum velocity (TAMXV) of the blood flow within the preovulatory follicle wall and the early corpus luteum. Ultrasonographic examinations began 48 h after a luteolytic injection of PGF(2alpha) analogue was given at the mid-luteal phase of the oestrous cycle. Cows with spontaneous ovulation were scanned at 6 h intervals until ovulation occurred. Cows with GnRH-induced ovulation were scanned just before GnRH injection (0 h), thereafter at 0.5, 1, 2, 6, 12, 24 h and at 24 h intervals up to day 5. Blood samples were collected at the same time points for oestradiol, LH and progesterone determinations. Cows with both spontaneous and GnRH-induced ovulation showed a clear increase in the plasma concentration of LH (LH surge) followed by ovulation 26-34 h later. In the colour Doppler image of the preovulatory follicle, the blood flow before the LH surge was detectable only in a small area in the base of the follicle. An acute increase in the blood flow velocity (TAMXV) was detected at 0.5 h after GnRH injection, synchronously with the initiation of the LH surge. At 12 h after the LH surge, the plasma concentrations of oestradiol decreased to basal concentrations. The TAMXV remained unchanged after the initial increase until ovulation, but decreased on day 2 (12-24 h after ovulation). In the early corpus luteum, the blood flow (area and TAMXV) gradually increased in parallel with the increase in corpus luteum volume and plasma progesterone concentration from day 2 to day 5, indicating active angiogenesis and normal luteal development. Collectively, the complex structural, secretory and functional changes that take place in the ovary before ovulation are closely associated with a local increase in the blood flow within the preovulatory follicle wall. The result of the present study provides the first visual information on vascular and blood flow changes associated with ovulation and early corpus luteum development in cows. This information may be essential for future studies involving pharmacological control of blood flow and alteration of ovarian function.     

Low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows

Objectives were to determine the effect of progesterone (P4) concentration on fertility of lactating dairy cows induced to ovulate follicles of the first follicular wave. Lactating dairy cows (n=989) at 38±3d postpartum were balanced by parity and body condition score and randomly assigned to 3 treatments: first follicular wave (FFW), first follicular wave with exogenous P4 (FFWP), or second follicular wave (SFW). All cows had their estrous cycle presynchronized with 2 injections of prostaglandin (PG) F(2α) given 14 d apart. Cows in the FFW and FFWP treatments started the ovulation synchronization protocol 3 d after the last PGF(2α) of the presynchronization protocol, whereas SFW cows received a GnRH injection (100 μg of gonadorelin diacetate; Cystorelin, Merial Ltd., Duluth, GA) 3 d after the last PGF(2α) of the presynchronization protocol and started the synchronization protocol 7 d later. The synchronization protocol consisted of GnRH on d -10, PGF(2α) on d -3, and GnRH concurrent with timed artificial insemination (AI) on d 0. Cows in the FFWP treatment received 2 controlled internal drug release inserts containing 1.38 g of P4 from d -8 to -3. Progesterone concentration was determined on d -10, -8, -6, -3, and 0 from all cows and at 7, 14, and 21 d after AI from a subsample of cows (n=170). Cows (n=715) had their ovaries scanned by ultrasound on d -10, -3, and 7 d. Pregnancy was diagnosed at 38 and 66 d after AI. Concentration of P4 from study d -8 to -3 was lowest for FFW cows (1.4±0.1 ng/mL) and similar between SFW (3.7±0.2 ng/mL) and FFWP (3.7±0.1 ng/mL) cows. Diameter of the dominant follicle on study d -3 was greater for FFW cows (16.5±0.3 mm) than for SFW cows (15.4±0.3 mm), but diameter of the dominant follicle of FFWP cows was not different (15.9±0.3 mm) compared with that of SFW and FFW cows. The incidence of multiple ovulation was largest for FFW cows (SFW=19.5, FFW=33.6, FFWP=19.0%), but pregnancy per AI (P/AI) at 66 d was smallest for FFW cows (SFW=38.9, FFW=22.3, FFWP=32.0%). Anovular cows in the SFW (19.4 vs. 42.8%) and FFWP (22.1 vs. 37.2%) treatments had reduced P/AI compared with cyclic cows, despite having similar or greater P4 concentration from study d -8 to -3, respectively. Estrus and ovulation synchronization protocols for lactating dairy cows must result in growth of ovulatory follicle under P4 concentration >2 ng/mL to ensure high P/AI.