M.M. Herlihy

Managing the dominant follicle in lactating dairy cows

Reproductive efficiency is not optimal in high-producing dairy cows. Although many aspects of ovarian follicular growth in cows are similar to those observed in heifers, there are numerous specific differences in follicular development that may be linked with changes in reproductive physiology in high-producing lactating dairy cows. These include: 1) reduced circulating estradiol (E2) concentrations near estrus, 2) ovulation of follicles that are larger than the optimal size, 3) increased double ovulation and twinning, and 4) increased incidence of anovulation with a distinctive pattern of follicle growth in anovular dairy cows. The first three changes become more dramatic as milk production increases, although anovulation has not generally been associated with level of milk production. To overcome reproductive inefficiencies in dairy cows, reproductive management programs have been developed to synchronize ovulation and enable the use of timed AI in lactating dairy cows. Effective regulation of the CL, follicles, and hormonal environment during each part of the protocol is critical for optimizing these programs. This review discusses the distinct aspects of follicular development in lactating dairy cows and the methodologies that have been utilized in the past two decades in order to manage the dominant follicle during synchronization of ovulation and timed AI programs.

Presynchronization with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating dairy cows

The objective of this study was to compare circulating progesterone (P4) profiles and pregnancies per AI (P/AI) in lactating dairy cows bred by timed artificial insemination (TAI) following Ovsynch-56 after 2 different presynchronization protocols: Double-Ovsynch (DO) or Presynch-Ovsynch (PS). Our main hypothesis was that DO would increase fertility in primiparous cows, but not in multiparous cows. Within each herd (n=3), lactating dairy cows (n=1,687; 778 primiparous, 909 multiparous) were randomly assigned to DO [n=837; GnRH-7d-PGF(2α)-3d-GnRH-7d-Ovsynch-56 (GnRH-7d-PGF(2α)-56h-GnRH-16hTAI)] or PS (n=850; PGF(2α)-14d-PGF(2α)-12d-Ovsynch-56). In 1 herd, concentrations of P4 were determined at the first GnRH (GnRH1) of Ovsynch-56 and at d 11 after TAI (n=739). In all herds, pregnancy was diagnosed by palpation per rectum at 39 d. In 1 herd, the incidence of late embryo loss was determined at 74d, and data were available on P/AI at the subsequent second service. Presynchronization with DO reduced the percentage of animals with low P4 concentrations (<0.50 ng/mL) at GnRH1 of Ovsynch-56 (5.4 vs. 25.3%, DO vs. PS). A lesser percentage of both primiparous and multiparous cows treated with DO had low P4 concentrations at GnRH1 of Ovsynch-56 (3.3 vs. 19.7%, DO vs. PS primiparous; and 8.8 vs. 31.9%, DO vs. PS multiparous). Presynchronization with DO improved P/AI at the first postpartum service (46.3 vs. 38.2%, DO vs. PS). Statistically, a fertility improvement could be detected for primiparous cows treated with DO (52.5 vs. 42.3%, DO vs. PS, primiparous), but only a tendency could be detected in multiparous cows (40.3 vs. 34.3%, DO vs. PS, multiparous), consistent with our original hypothesis. Presynchronization treatment had no effect on the incidence of late embryo loss after first service (8.5 vs. 5.5%, DO vs. PS). A lower body condition score increased the percentage of cows with low P4 at GnRH1 of Ovsynch-56 and reduced fertility to the TAI. In addition, P4 concentration at d 11 after TAI was reduced by DO. The method of presynchronization at first service had no effect on P/AI at the subsequent second service (34.7 vs. 36.5%, DO vs. PS). Thus, presynchronization with DO induced cyclicity in most anovular cows and improved fertility compared with PS, suggesting that DO could be a useful reproductive management protocol for synchronizing first service in commercial dairy herds.

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.

Relationships between fertility and postpartum changes in body condition and body weight in lactating dairy cows

The relationship between energy status and fertility in dairy cattle was retrospectively analyzed by comparing fertility with body condition score (BCS) near artificial insemination (AI; experiment 1), early postpartum changes in BCS (experiment 2), and postpartum changes in body weight (BW; experiment 3). To reduce the effect of cyclicity status, all cows were synchronized with Double-Ovsynch protocol before timed AI. In experiment 1, BCS of lactating dairy cows (n = 1,103) was evaluated near AI. Most cows (93%) were cycling at initiation of the breeding Ovsynch protocol (first GnRH injection). A lower percentage pregnant to AI (P/AI) was found in cows with lower (≤ 2.50) versus higher (≥ 2.75) BCS (40.4 vs. 49.2%). In experiment 2, lactating dairy cows on 2 commercial dairies (n = 1,887) were divided by BCS change from calving until the third week postpartum. Overall, P/AI at 70-d pregnancy diagnosis differed dramatically by BCS change and was least for cows that lost BCS, intermediate for cows that maintained BCS, and greatest for cows that gained BCS [22.8% (180/789), 36.0% (243/675), and 78.3% (331/423), respectively]. Surprisingly, a difference existed between farms with BCS change dramatically affecting P/AI on one farm and no effect on the other farm. In experiment 3, lactating dairy cows (n = 71) had BW measured weekly from the first to ninth week postpartum and then had superovulation induced using a modified Double-Ovsynch protocol. Cows were divided into quartiles (Q) by percentage of BW change (Q1 = least change; Q4 = most change) from calving until the third week postpartum. No effect was detected of quartile on number of ovulations, total embryos collected, or percentage of oocytes that were fertilized; however, the percentage of fertilized oocytes that were transferable embryos was greater for cows in Q1, Q2, and Q3 than Q4 (83.8, 75.2, 82.6, and 53.2%, respectively). In addition, percentage of degenerated embryos was least for cows in Q1, Q2, and Q3 and greatest for Q4 (9.6, 14.5, 12.6, and 35.2% respectively). In conclusion, for cows synchronized with a Double-Ovsynch protocol, an effect of low BCS (≤ 2.50) near AI on fertility was detected, but change in BCS during the first 3 wk postpartum had a more profound effect on P/AI to first timed AI. This effect could be partially explained by the reduction in embryo quality and increase in degenerate embryos byd 7 after AI in cows that lost more BW from the first to third week postpartum.

Effect of timing of initiation of resynchronization and presynchronization with gonadotropin-releasing hormone on fertility of resynchronized inseminations in lactating dairy cows.

Lactating Holstein cows (n=1,456) were randomized in a 2×2 factorial design to compare the main effects of day of initiation of resynchronization after artificial insemination (AI; 32 vs. 39 d) and presynchronization with GnRH 7d before initiation of resynchronization on fertility to timed AI (TAI). This design resulted in the following 4 resynchronization treatments: (1) resynchronization (GnRH treatment, PGF2α treatment 7d later, GnRH treatment 56 h later, and TAI 16 h later), initiated 32 ± 3 d after AI; (2) presynchronization with 100 µg of GnRH 25 ± 3 d after AI and resynchronization initiated 32 ± 3 d after AI at nonpregnancy diagnosis; (3) resynchronization initiated 39 ± 3 d after AI (GPG39); and (4) presynchronization with 100 µg of GnRH 32 ± 3 d after AI at nonpregnancy diagnosis and resynchronization initiated 39 ± 3 d after AI. Overall, 344 cows were inseminated at estrus between enrollment (25 ± 3 d after AI) and TAI of the resynchronization treatments, and 1,112 cows received TAI. Progesterone (P4) was analyzed in blood samples collected from all cows at the first GnRH injection of the resynchronization protocols (G1), and ovarian structures were evaluated and blood samples were collected at G1, at the PGF2α injection, and at the TAI of the resynchronization protocols in a subgroup of cows (n=417). When analyzed as main effects, cows presynchronized with GnRH had more pregnancies per AI (P/AI) than nonpresynchronized cows (38.9 vs. 33.8%), whereas timing of initiation of resynchronization did not affect P/AI. Although cows with high P4 at G1 had greater P/AI than cows with low P4 (38.7 vs. 31.8%), presynchronization with GnRH did not increase the proportion of cows with high P4 (>1.0 ng/mL) at G1 but moved cows from a low-P4 environment to an intermediate-P4 level. Presynchronization with GnRH also decreased the percentage of cows with low P4 at the PGF2α injection, thereby increasing synchrony to the protocol. Cows with high P4 at G1 had a decreased ovulatory response to G1 compared with cows with low P4 (40.9 vs. 69.1%), and cows that ovulated to G1 had decreased luteal regression after PGF2α compared with cows that did not ovulate (78.5 vs. 87.3%). We conclude that presynchronization with GnRH 7d before initiation of resynchronization increased fertility in dairy cows, whereas timing of initiation of resynchronization did not.

Effect of presynchronization with human chorionic gonadotropin or gonadotropin-releasing hormone 7 days before resynchronization of ovulation on fertility in lactating dairy cows

Our objectives were to (1) compare the effect on pregnancies per artificial insemination (P/AI) of presynchronization of the estrous cycle with human chorionic gonadotropin (hCG) 7d before resynchronization of ovulation (Resynch) initiated 25 d after timed artificial insemination (TAI) and compare the presynchronization treatment with the Double-Ovsynch (DO) protocol, and (2) evaluate whether hCG for presynchronization could be replaced with GnRH. In experiment 1, lactating Holstein cows were blocked by parity and were randomly assigned to receive (1) Resynch-25 (D25), the Resynch protocol (GnRH-7 d-PGF(2 α)-56 h-GnRH-16 h-TAI) initiated 25 d after TAI (n=418); (2) HGPG, presynchronization with hCG (2,000 IU of Chorulon) 7d before D25 (n=450); and (3) DO (Pre-Resynch, GnRH-7 d-PGF(2 α)-72 h-GnRH; Breeding-Resynch, GnRH-7 d-PGF(2 α)-56 h-GnRH-16 h-TAI) initiated 22d after TAI (n=405). At 29 d after TAI, cows in the HGPG (37.3%) and DO (35.8%) groups had more P/AI than did cows in the D25 group (28.0%), and cows in the HGPG and DO groups continued to have more P/AI than did cows in the D25 group at 53 d after TAI. Presynchronization with hCG induced ovulation in 76% of the cows, which increased the percentage of HGPG cows with a corpus luteum at the initiation of Resynch compared with cows in the D25 group. In experiment 2, the D25 (n=368) and HGPG (n=338) treatments described in experiment 1 were compared in addition to a third treatment (GGPG; n=351), in which the hCG injection 18 d after TAI was replaced with a GnRH injection (200 µg of gonadorelin). At 32 d after TAI, cows in the HGPG group had more P/AI than did cows in the D25 group (33.7 vs. 25.5%), whereas cows in the GGPG group had intermediate P/AI (31.6%). At 53 d after TAI, P/AI tended to be greater for cows in the HGPG group than for those in the D25 group, whereas P/AI for cows in the GGPG group did not differ from that for cows in the D25 group. Treatment with hCG and GnRH 18d after TAI induced ovulation in 58.8 and 48.2% of cows, respectively, but did not increase the percentage of cows with a corpus luteum at the initiation of Resynch. More cows in the HGPG and GGPG groups had their estrous cycles synchronized after the resynchronization protocols compared with cows in the D25 group. We conclude that presynchronization with hCG increased fertility by increasing synchronization to the Resynch protocol, whereas presynchronization with GnRH improved synchronization to the Resynch protocol but did not improve fertility when compared with no presynchronization or presynchronization with hCG.

Evaluation of protocols to synchronize estrus and ovulation in seasonal calving pasture-based dairy production systems

Lactating dairy cows (n=1,538) were enrolled in a randomized complete block design study to evaluate protocols to synchronize estrus and ovulation. Within each herd (n=8), cows were divided into 3 calving groups: early, mid, and late, based on days in milk (DIM) at mating start date (MSD). Early calving cows (n=1,244) were ≥42 DIM at MSD, mid-calving cows (n=179) were 21 to 41 DIM at MSD, and late-calving cows (n=115) were 0 to 20 DIM at MSD. Cows in the early, mid-, and late-calving groups were synchronized to facilitate estrus or timed AI (TAI) at MSD (planned breeding 1; PB1), 21 d (PB2), and 42 d (PB3) after MSD, respectively. For each PB, cows in the relevant calving group were stratified by parity and calving date and randomly assigned to 1 of 4 experimental groups: (1) d -10 GnRH (10 μg of i.m. buserelin) and controlled internal drug release insert (CIDR; 1.38 g of progesterone); d -3 PGF(2α) (25 mg of i.m. dinoprost); and d -2 CIDR out and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH 36 h after CIDR out and TAI 18 h later (CIDR_TAI); (3) same as CIDR_TAI, but no CIDR (Ovsynch); or (4) untreated controls (CTRL). The CIDR_OBS, CIDR_TAI, and Ovsynch had shorter mean intervals from calving to first service compared with the CTRL (69.2, 63.4, and 63.7 vs. 73.7 d, respectively). Both CIDR_OBS (predicted probability; PP of pregnancy=0.59) and CIDR_TAI (PP of pregnancy=0.54) had increased odds of conceiving at first service compared with Ovsynch [PP of pregnancy=0.45; odds ratio (OR)=1.81 and OR=1.46, respectively], and Ovsynch had decreased likelihood of conceiving at first service (OR=0.70) compared with CTRL (PP of pregnancy=0.53). Both CIDR_TAI hazard ratio; HR [95% confidence interval=1.21 (1.04, 1.41)] and Ovsynch [HR (95% confidence interval)=1.23 (1.05, 1.44)] were associated with an increased likelihood of earlier conception compared with the CTRL. A greater proportion of cows on the CIDR_TAI treatment successfully established pregnancy in the first 42 d of the breeding season compared with the CTRL (0.75 vs. 0.67 PP of 42-d pregnancy, respectively). Protocols to synchronize estrus and ovulation were effective at achieving earlier first service and conception in pasture-based seasonal calving dairy herds. However, animals that conceived following insemination at observed estrus had a decreased likelihood of embryo loss to first service compared with animals bred with TAI (PP of embryo loss after first service=0.05 vs. 0.09; OR=0.52).

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.

Factors associated with fertility outcomes in cows treated with protocols to synchronize estrus and ovulation in seasonal-calving, pasture-based dairy production systems.

Logistic regression was used to identify factors associated with fertility outcomes in cows treated with protocols to synchronize estrus and ovulation. Lactating dairy cows (n=1,538) were enrolled in a completely randomized block design study to evaluate synchronization treatments. Within each herd (n=8), cows were divided into 3 calving groups: early [≥ 42 d in milk (DIM) at mating start date (MSD); n=1,244], mid (21 to 41 DIM at MSD; n=179), and late (0 to 20 DIM at MSD; n=115), based on DIM at MSD. Cows in the early-, mid-, and late-calving groups were synchronized to facilitate estrus or timed artificial insemination (TAI) at MSD (planned breeding 1; PB1), 21 d (PB2), and 42 d (PB3) after MSD, respectively. For each PB, cows in the relevant calving group were stratified by parity and calving date and randomly assigned to (1) d -10 GnRH (10 μg i.m. of buserelin) and CIDR [controlled internal drug release insert, 1.38 g of progesterone (P4)]; d -3 PGF(2α) (25mg i.m. of dinoprost); d -2 CIDR out and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH 36 h after CIDR out and TAI 18 h later (CIDR_TAI); (3) same as CIDR_TAI, but no CIDR (i.e., Ovsynch); or (4) untreated controls (CTRL). Use of a CIDR-based ovulation synchronization protocol (i.e., CIDR_TAI) increased synchronization rates in anovular cows. Both CIDR_OBS and CIDR_TAI animals without a corpus luteum (CL) had increased likelihood of conception at first service compared with Ovsynch animals without a CL. Animals with low body condition score (BCS) treated with CIDR_OBS had an increased likelihood of conceiving at first service compared with low-BCS animals treated with CIDR_TAI, Ovsynch, or CTRL. Animals <60 d in milk (DIM) treated with CIDR_OBS and CIDR_TAI had increased likelihood of conceiving at first service compared with animals treated with Ovsynch. Treatment with CIDR_TAI increased synchronization rate in cows categorized as low BCS, anovulatory, and <60 DIM compared with both CIDR_OBS and Ovsynch, and increased submission rate compared with CIDR_OBS. Conception rate in cows within these categories, however, was greatest for CIDR_OBS, resulting in minimal differences in actual pregnancy rates between CIDR_OBS and CIDR_TAI treatments, both of which were superior to Ovsynch. Treatment differences in the response variables investigated were minimal in cows categorized as medium or high BCS, ovulatory, and >60 DIM, indicating that CIDR-based protocols could be targeted at particular cows, and all other cows could be synchronized using Ovsynch.

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine parameters in seasonal-calving, pasture-based Holstein-Friesian cows

Fertility to timed AI (TAI) is profoundly affected by progesterone (P4) levels during hormonal synchronization protocols. Holstein-Friesian dairy cows managed in a seasonal-calving, pasture-based production system were randomly assigned to 2 treatments to manipulate P4 before TAI during growth of the preovulatory follicle. Cows in the first treatment (High P4; n=30) were submitted to a Double-Ovsynch protocol {Pre-Ovsynch [GnRH; 7 d, PGF2α; 3 d, GnRH] followed 7 d later by Breeding-Ovsynch [GnRH (G1); 7 d PGF2α; 24 h, PGF2α; 32 h, GnRH (G2); 16 h, TAI]}. Cows in the second treatment (n=30; Low P4) received the same Double-Ovsynch protocol but with an additional PGF2α treatment 24 h after G1. Overall, synchronization rate did not differ between treatments and was 92% (55/60). Unexpectedly, 37% of Low P4 cows were detected in estrus ~24 h before scheduled TAI and were inseminated ~16 h before scheduled TAI. Overall, P4 did not differ between treatments at G1, whereas High P4 cows had greater P4 concentrations at PGF2α and G2 than Low P4 cows. High P4 cows had the smallest mean follicle diameter at G2, whereas Low P4 cows with no estrus before TAI had intermediate mean follicle diameter at G2, and Low P4 cows with estrus before TAI had the largest mean follicle diameter. Low P4 cows with estrus before TAI had larger corpora lutea 15 d after TAI than Low P4 cows without estrus before TAI or High P4 cows. In accordance with corpus luteum size on d 15, High P4 cows and Low P4 cows without estrus before TAI had lower P4 from 4 to 46 d after TAI than Low P4 cows with estrus before TAI. Relative mRNA levels of the interferon-stimulated genes ISG15, MX1, MX2, and OAS1 were greater for Low P4 than for High P4 cows, whereas relative mRNA levels of RTP4 were greater for High P4 than for Low P4 cows 18 d after TAI. Treatment did not affect plasma pregnancy-associated glycoprotein concentrations after TAI; however, pregnancy-associated glycoprotein concentrations were affected by pregnancy status and parity. Treatment did not affect pregnancy per artificial insemination at 29, 39, or 60 d after TAI, and no pregnancy losses were observed from 39 to 60 d after TAI. We concluded that (1) Low P4 cows were more likely to express estrus than High P4 cows; (2) the subpopulation of Low P4 cows that expressed estrus had larger preovulatory follicles and greater P4 concentrations after TAI; and (3) regardless of estrus before TAI, all Low P4 cows had greater mRNA expression for 5 of 6 interferon-stimulated genes than High P4 cows 18 d after TAI.