Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous Holstein cows

Our objective was to evaluate the effect of manipulating progesterone (P4) concentrations before timed artificial insemination (TAI) on reproductive and endocrine outcomes in high-producing Holstein cows. Multiparous lactating Holstein cows (n = 80) were synchronized for first TAI using a Double-Ovsynch protocol and were randomly assigned to receive 25 mg of PGF_2α 1 d after the first GnRH treatment of the Breeding-Ovsynch protocol that included a once-used P4 insert (low-P4 group) or to receive 2 new P4 inserts during the Breeding-Ovsynch protocol (high-P4 group). Blood samples were collected thrice weekly from ?10 to 32 d relative to TAI for all cows and from 32 to 67 d after TAI for pregnant cows and were analyzed for P4 and pregnancy-specific protein B (PSPB) concentrations. Expression of IFNτ-stimulated gene 15 (ISG15) was assessed in blood leukocytes 18 and 20 d after TAI. As expected, P4 concentrations were greater for high-P4 cows than for low-P4 cows from 3 to 8 d before TAI. Incidence of double ovulation was 3-fold greater for low-P4 cows than for high-P4 cows (33 vs. 10%), which resulted in more twin pregnancies 32 d after TAI for low-P4 cows than for high-P4 cows (29 vs. 0%). Low-P4 cows had larger preovulatory follicles at the last GnRH treatment of the Double-Ovsynch protocol and greater P4 concentrations than high-P4 cows after TAI. Relative expression of ISG15 mRNA 18 and 20 d after TAI was greater for low-P4 cows than for high-P4 cows and for pregnant cows than for nonpregnant cows. Overall, PSPB concentrations tended to be greater for low-P4 cows than for high-P4 cows, and pregnant cows had greater P4 concentrations than nonpregnant cows. In summary, cows with low P4 before TAI had increased preovulatory follicle diameter, PSPB concentrations, relative expression of ISG15 mRNA 18 and 20 d after TAI, double ovulations, and twinning compared with cows with high P4 before TAI. Increasing P4 before TAI may effectively decrease double ovulation and twinning in high-producing multiparous Holstein cows.     

Characterization of luteal dynamics in lactating Holstein cows for 32 days after synchronization of ovulation and timed artificial insemination

Approximately 20 to 30% of cows diagnosed not pregnant 32 d after timed artificial insemination (TAI) lack a corpus luteum (CL), and cows submitted to a resynchronization protocol in the absence of a CL have about 10% fewer pregnancies per AI (P/AI) than cows with a CL. An understanding of luteal dynamics after synchronization of ovulation and TAI may help refine strategies for reinseminating cows failing to conceive. Lactating Holstein cows (n = 141) were synchronized for first TAI using a Double-Ovsynch protocol. Thrice weekly from 4 to 32 d after TAI, blood samples were collected for evaluation of plasma progesterone (P4) concentrations, and CL diameter was measured using transrectal ultrasonography. Pregnancy status was determined using transrectal ultrasonography 32 d after TAI. Nonsynchronized cows (n = 4) were removed from the study. For cows diagnosed pregnant 32 d after TAI (n = 57), P4 increased from 4 to 15 d and then remained constant until 32 d after TAI, whereas CL volume increased from 4 to 11 d and then remained constant until 32 d after TAI. For cows diagnosed not pregnant 32 d after TAI (n = 80), P4 profiles were evaluated using statistical cluster analysis based on the day after TAI that P4 decreased to <1 ng/mL, resulting in 5 clusters: (1) CL regression 15 d after TAI (1.3%), (2) CL regression 18 to 22 d after TAI (55.0%), (3) CL regression 25 to 27 d after TAI (17.5%), (4) CL regression 29 to 32 d after TAI (5.0%), and (5) CL maintained until 32 d after TAI (21.3%). Plasma pregnancy-associated glycoprotein (PAG) levels at 25 and 32 d after TAI differed among clusters and were below the cut-off value of the assay for the classification of cows as not pregnant for cows in clusters 2, 3, and 4, whereas more than half of the cows in cluster 5 had increased plasma PAG levels. We conclude that at least half of the nonpregnant cows that maintained their CL until 32 d after TAI were initially pregnant but underwent early pregnancy loss based on increased plasma PAG levels at 25 and 32 d after TAI.     

A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.     

Effect of interval to resynchronization of ovulation on fertility of lactating Holstein cows when using transrectal ultrasonography or a pregnancy-associated glycoprotein enzyme-linked immunosorbent assay to diagnose pregnancy status

The objective of this study was to compare 2 strategies for resynchronization of ovulation based on nonpregnant diagnoses using transrectal ultrasonography or a pregnancy-associated glycoprotein (PAG) ELISA. Lactating Holstein cows (n = 1,038) were submitted for first postpartum timed artificial insemination (TAI) using a Presynch + Ovsynch protocol. After the initial breeding, cows were randomly assigned to initiate resynchronization 25 d (D25) or 32 d (D32) later. Pregnancy status of cows initiating Resynch 25 d after TAI was determined 27 d after TAI by using a PAG ELISA, whereas pregnancy status of cows initiating Resynch 32 d after TAI was determined 39 d after TAI using transrectal ultrasonography. Cows diagnosed as not pregnant continued the Resynch protocol by receiving an injection of PGF_2α 7 d after the initial GnRH injection and a second GnRH injection 54 h after the PGF_2α injection. Cows in both treatments were inseminated approximately 16 h after the second GnRH injection. Blood samples for analysis of progesterone (P₄) were collected at the first GnRH injection of each Resynch protocol. Pregnancies per AI (P/AI) of nonpregnant cows initiating Resynch 25 vs. 32 d after first postpartum TAI did not differ 39 d after TAI and were 28.3 vs. 30.9% for D25 vs. D32 cows, respectively. Mean P₄ at the first GnRH injection of Resynch was greater for D32 than for D25 cows (3.67 ± 0.22 vs. 2.83 ± 0.22 ng/mL), indicating that the Resynch treatments were initiated at different stages of the estrous cycle. After blocking P₄ concentration into low (<1.0 ng/mL) or high (≥1.0 ng/mL) classes, P₄ class was not found to affect P/AI 39 d after TAI. Early resynchronization was not found to affect P/AI 39 d after TAI; however, early resynchronization did decrease days between inseminations and the interval from the initial nonpregnant diagnosis to conception. Earlier detection of nonpregnant cows using the PAG ELISA in conjunction with a TAI resynchronization program may improve the rate at which cows become pregnant in a dairy herd compared with transrectal ultrasonography conducted at a later stage after TAI.     

Temporarily decreasing progesterone after timed artificial insemination decreased expression of interferon-tau stimulated gene 15 (ISG15) in blood leukocytes,serum pregnancy-specific protein B concentrations,and embryo size in lactating Holstein cows

Our objective was to evaluate the effects of temporarily decreasing progesterone (P4) after timed artificial insemination (TAI) on embryonic growth in dairy cows. Lactating Holstein cows (n = 80) were submitted to a Double-Ovsynch protocol for first TAI and were assigned randomly to receive 12.5 mg of PGF_2α 5 d after the last GnRH treatment (LowP4) or remain untreated (control). Blood samples were collected thrice weekly from 5 to 29 d after TAI for all cows and from 32 to 67 d for pregnant cows, and were analyzed for P4 and pregnancy-specific protein B concentrations. Expression of interferon-tau stimulated gene 15 (ISG15) was assessed in blood leukocyte mRNA 18 and 20 d after TAI. Pregnancy diagnosis was performed weekly using ultrasound from 32 to 67 d after TAI, and embryonic crown-rump length was measured 32, 39, and 46 d after TAI. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures of SAS. The LowP4 cows had less P4 than control cows from 6 to 11 d after TAI; however, pregnancy outcomes 32 d after TAI and pregnancy loss from 32 to 67 d after TAI did not differ between treatments. Control cows diagnosed pregnant 32 d after TAI had greater expression of ISG15 20 d after TAI than LowP4 cows diagnosed pregnant 32 d after TAI, and pregnant control cows had greater pregnancy-specific protein B concentrations from 25 to 67 d after TAI than pregnant LowP4 cows. Embryo size did not differ between treatments 32 and 39 d after TAI, but control cows had larger embryos 46 d after TAI. In conclusion, temporarily decreasing P4 after TAI decreased embryonic growth during early pregnancy in lactating Holstein cows but did not affect pregnancies per artificial insemination or pregnancy loss.     

Pregnancy rates in lactating dairy cows after presynchronization of estrous cycles and variations of the Ovsynch protocol

Our objectives were to determine pregnancy rates after altering times of the second GnRH injection, insemination, or both in a combined Presynch + Ovsynch protocol, to accommodate once-daily lockup of dairy cows. Lactating dairy cows (n = 665) from 2 dairy herds in northeastern Kansas were studied. Cows ranged from 24 to 44 d in milk (DIM) at the start of the Pre-synch protocol, which consisted of 2 injections of PGF(2alpha) 14 d apart, with the second injection given 12 d before initiating the Ovsynch protocol. Cows were blocked by lactation number and assigned randomly to 3 treatments consisting of variations of the Ovsynch protocol. Cows in 2 treatments received injections of GnRH 7 d before and 48 h (G48) after the PGF(2alpha) injection. Timed AI (TAI) was conducted at the time of the second GnRH injection (G48 + TAI48) or 24 h later (G48 + TAI72). Cows in the third treatment received the injections of GnRH 7 d before and at 72 h after PGF(2alpha) and were inseminated at the time of the second GnRH injection (G72 + TAI72). Pregnancy was diagnosed weekly by palpation per rectum of uterine contents on d 40 or 41 after TAI. Pregnancy rates differed between herds, but they were consistently greater for G72 + TAI72 than for G48 + TAI48 and G72 + TAI72. Subsequent calving rates were consistent with differences in initial TAI pregnancy rates. Pregnancy loss was least for cows on the G72 + TAI72 treatment. Body condition scores (BCS) ranged from 1.0 to 4.0 when assessed on Monday of the breeding week. An interaction of BCS and herd was detected in which cows in herd 1 having poorer BCS (<2.25) had greater pregnancy rates than cows of greater BCS (>/=2.25), whereas the reverse was true in herd 2 in which overall pregnancy rates were greater. We concluded that inseminating at 48 or 72 h after PGF(2alpha), when GnRH was administered at 48 h after PGF(2alpha), produced fewer pregnancies than inseminating and injecting GnRH at 72 h after PGF(2alpha) for cows whose estrous cycles were synchronized before initiating this variant of the Ovsynch protocol.     

Short communication: Presynchronization for timed artificial insemination in grazing dairy cows by using progesterone for 14 days with or without prostaglandin F(2α) at the time of progesterone withdrawal

Progesterone-containing devices can be inserted intravaginally for 14 d to presynchronize the estrous cycle for timed artificial insemination (TAI) in beef heifers ("14-day CIDR-PG" or "Show-Me-Synch" program). The progesterone treatment is effective for presynchronization because cattle develop a persistent dominant follicle during treatment that ovulates within 3 d after progesterone removal. The subsequent estrous cycle can be effectively used for a TAI program. Some cattle will retain a functional corpus luteum (CL) for the entire 14-d treatment period and will not be synchronized effectively because the interval to ovulation depends on the lifespan of their existing CL. The objective was to test the effect of a luteolytic dose of PGF(2α) at progesterone removal for improving synchrony of estrus after treatment and increasing conception rate to a subsequent TAI in dairy cows. Postpartum cows (n = 1,021) from 2 grazing dairy herds were assigned to 1 of 2 presynchronization programs that used a controlled internal drug releasing (CIDR) device containing progesterone: 14dCIDR (CIDR in, 14 d, CIDR out; n = 523) or 14dCIDR+PGF(2α) (CIDR in, 14 d, CIDR out, and PGF(2α); n = 498). Cows were body condition scored (BCS; 1 to 5, thin to fat) and tail painted at CIDR removal. Paint score (PS) was recorded after CIDR removal [PS = 0 (all paint removed, indication of estrus), PS = 3 (paint partially removed), or PS = 5 (no paint removed; indication of no estrus)]. At 19 d after CIDR removal, all cows were treated with PGF(2α), 56 h later treated with GnRH, and then 16 h later were TAI. Treating cows with PGF(2α) at CIDR removal increased the percentage with PS = 0 within 5 d (58.1% vs. 68.9%; 14dCIDR vs. 14dCIDR+PGF(2α)). We found no effect of treatment, however, on conception rate at TAI (41.1% vs. 43.6%; respectively). The TAI conception rate increased with increasing BCS and was greater for cows that had PS = 0 within 5 d after CIDR removal. In summary, treating cows with PGF(2α) at CIDR removal increased the percentage of cows with all tail paint removed but did not increase percentage of pregnant cows after TAI.     

Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows

Objectives were to develop a timed artificial insemination (TAI) resynchronization program to improve pregnancy per AI and to evaluate responses of circulating progesterone and pregnancy-associated glycoproteins in lactating cows. Cows (n = 1,578) were presynchronized with 2 injections of PGF_2α, given 14 d apart starting on d 45 ± 3 postpartum, followed by Ovsynch [2 injections of GnRH 7 d before and 56 h after injection of PGF_2α, TAI 16 h after second injection (d 0)]. The Resynch-treated cows received an intravaginal progesterone insert from d 18 to 25, GnRH on d 25, and pregnancy diagnosis on d 32, and nonpregnant cows received PGF_2α., GnRH 56 h later, and TAI 16 h later (d 35). The control cows were diagnosed for pregnancy on d 32 and nonpregnant cows received GnRH, PGF_2α 39 d after TAI, GnRH 56 h later, and TAI 16 h later (d 42). Pregnancy was reconfirmed on d 60 after AI. Ovarian structures were examined in a subset of cows at the time of GnRH and PGF_2α injections. Blood samples for analyses of progesterone and pregnancy-associated glycoproteins were collected every 2 d from d 18 to 30 in 100 cows, and collection continued weekly to d 60 for pregnant cows (n = 43). Preenrollment pregnancies per AI on d 32 did not differ for cows subsequently treated as Resynch (45.8%, n = 814) and control (45.9%, n = 764), and pregnancy losses on d 60 were 6.7 and 4.0%, respectively. Resynchronized service pregnancy per AI (36%, n = 441; 39.5%, n = 412) and pregnancy losses (6.3 and 6.7%) did not differ for Resynch and control treatments, respectively. Days open for pregnant cows after 2 TAI were less for the Resynch treatment than for the control treatment (96.2 ± 0.82 vs. 99.5 ± 0.83 d). Cows in the Resynch treatment had more large follicles at the time of GnRH. The number of corpora lutea did not differ between treatments at the time of PGF_2α. Plasma progesterone for pregnant cows was greater for Resynch cows than for control cows (18-60 d; 6.6 vs. 5.3 ng/mL), and plasma concentrations of progesterone on d 18 were greater for pregnant cows than for nonpregnant cows (5.3 vs. 4.3 ng/mL). Plasma pregnancy-associated glycoproteins during pregnancy were lower for cows in the Resynch treatment compared with control cows on d 39 (2.8 vs. 4.1 ng/mL) and 46 (1.3 vs. 3.0 ng/mL). Cows pregnant on d 32 that lost pregnancy by d 60 (n = 7) had lower plasma concentrations of pregnancy-associated glycoproteins on d 30 than cows that maintained pregnancy (n = 36; 2.9 vs. 5.0 ng/mL). Pregnancy-associated glycoproteins on d 30 (>0.33 ng/mL) were predictive of a positive d 32 pregnancy diagnosis (sensitivity = 100%; specificity = 90.6%). In conclusion, Resynch and control protocols had comparable pregnancy per AI for first and second TAI services, but pregnancy occurred 3.2 d earlier in the Resynch group because inseminations in the Resynch treatment began 7 d before those in the control treatment. Administration of an intravaginal progesterone insert, or GnRH, or both increased progesterone during pregnancy. Dynamics of pregnancy-associated glycoproteins were indicative of pregnancy status and pregnancy loss.     

Fertility of dairy cows after resynchronization of ovulation at three intervals following first timed insemination

Lactating Holstein cows (n = 711) on a commercial dairy farm in Wisconsin received a hormonal synchronization protocol to initiate first timed artificial insemination (TAI) on the following postpartum schedule: two injections of 25 mg PGF2alpha at 32 +/- 3 d and 46 +/- 3 d (Presynch); 100 microg GnRH at 60 +/- 3 d; 25 mg PGF2alpha at 67 +/- 3 d; and 100 microg GnRH + TAI at 69 +/- 3 d (Ovsynch). At first TAI, cows were randomly assigned to initiate the first GnRH injection of a hormonal protocol for resynchronization of ovulation (Resynch; 100 microg GnRH, d 0, 25 mg PGF2alpha, d 7, 100 microg GnRH + TAI, d 9) at 19 (D19), 26 (D26), or 33 d (D33) after first TAI to set up a second TAI service for cows failing to conceive to Ovsynch. Overall pregnancy rate per artificial insemination (PR/AI) to Ovsynch assessed 68 d after TAI was 31% and did not differ among treatment groups. For Resynch, PR/AI was assessed 26 d after TAI for D19 and D26 cows and 33 d after TAI for D33 cows. Overall PR/AI to Resynch was 32%. However, the PR/AI for D26 (34%) and D33 (38%) cows to Resynch was greater than for D19 cows (23%). Cows with a CL at the PGF2alpha injection (D19 cows) or at the first GnRH injection (D26 + D33 cows) of Resynch exhibited greater PR/AI to Resynch compared with cows without a CL. Survival analysis (failure time) of cows in the D26 and D33 treatment groups across the first three TAI services did not differ statistically. Although administration of GnRH to pregnant cows 19 d after first TAI service did not appear to induce iatrogenic embryonic loss, initiation of Resynch 19 d after first TAI service resulted in a lower PR/AI compared with initiation of Resynch 26 or 33 d after first TAI service.     

Combined use of Ovsynch and progesterone supplementation after artificial insemination in dairy cattle

The objective of this study was to evaluate the effects of different Ovsynch protocols combined with progesterone (P4) supplementation after artificial insemination (AI) of Holstein-Friesian cows. Cows were randomly synchronized at 52 to 63 d after parturition with either the classical Ovsynch protocol (GnRH on d 0, PGF(2α) on d 7, GnRH 48 h after PGF(2α)) or with a modified Ovsynch protocol (second GnRH 60 h after PGF(2α)). On d 4 after timed AI (TAI), the cows were blocked by parity and randomly divided into 2 groups. Half of the cows were supplemented with P4 (P4+) by applying a P4-releasing intravaginal device intravaginally for 14 d, whereas the other half remained untreated (P4-). In 50% of randomly chosen cows, plasma P4 was measured on d 4, 5, and 18 after TAI. Sonographic pregnancy diagnosis was performed on d 33 after TAI in a total of 398 cows. Health status and body condition score (BCS) of all cows were examined at several stages of the study. Cows in the modified Ovsynch protocol tended to have higher P4 values on d 4 after TAI than cows in the classical Ovsynch protocol (2.1 ± 0.2 vs. 1.6 ± 0.2 ng/mL), but no difference in pregnancy per AI (P/AI) was observed between the 2 Ovsynch protocols (38.4% vs. 44.1%). Independent of the Ovsynch protocols, P4+ cows tended to have higher P/AI compared with P4- cows (44.4% vs. 38.1%). The retention of fetal membranes and BCS at the time of insemination affected P/AI. Moreover, an interaction between BCS at the time of insemination and P4 supplementation was apparent; that is, the difference in P/AI between P4+ and P4- cows was significant in cows with BCS ≥3.25. Progesterone-supplemented cows showed higher P4 values on d 5 (4.9 ± 0.2 vs. 2.6 ± 0.2) and d 18 (7.8 ± 0.2 vs. 6.3 ± 0.2) after TAI, respectively. In conclusion, the elongation of the time interval between the injections of PGF(2α) and the second GnRH from 48 to 60 h had no effect on P/AI. Progesterone supplementation after insemination improved the P/AI of the Ovsynch protocols, but this effect was more apparent in cows with BCS ≥3.25.     

Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial insemination

To determine the accuracy of a pregnancy-associated glycoprotein (PAG) ELISA in identifying pregnancy status 27 d after timed artificial insemination (TAI), blood samples were collected from lactating Holstein cows (n = 1,079) 27 d after their first, second, and third postpartum TAI services. Pregnancy diagnosis by transrectal ultrasonography (TU) was performed immediately after blood sample collection, and pregnancy outcomes by TU served as a standard to test the accuracy of the PAG ELISA. Pregnancy outcomes based on the PAG ELISA and TU that agreed were considered correct, whereas the pregnancy status of cows in which pregnancy outcomes between PAG and TU disagreed were reassessed by TU 5 d later. The accuracy of pregnancy diagnosis was less than expected when using TU 27 d after TAI (93.7 to 97.8%), especially when pregnancy outcomes were based on visualization of chorioallantoic fluid and a corpus luteum but when an embryo was not visualized. The accuracy of PAG ELISA outcomes 27 d after TAI was 93.7, 95.4, and 96.2% for first, second, and third postpartum TAI services, respectively. Statistical agreement (kappa) between TU and the PAG ELISA 27 d after TAI was 0.87 to 0.90. Pregnancy outcomes based on the PAG ELISA had a high negative predictive value, indicating that the probability of incorrectly administering PGF_2α to pregnant cows would be low if this test were implemented on a commercial dairy.     

Effect of interval from timed artificial insemination to initiation of resynchronization of ovulation on fertility of lactating dairy cows

To compare 2 strategies for systematically resynchronizing ovulation, lactating Holstein cows (n = 763) at various days in milk and prior artificial insemination services were assigned randomly at timed AI (TAI) to receive the first GnRH injection of Ovsynch 26 (D26) or 33 (D33) d after TAI to resynchronize ovulation (Resynch) in cows failing to conceive. Cows in the D26 treatment received GnRH 26 d after TAI and continued Resynch only when diagnosed not pregnant by using ultrasonography 33 d after TAI, whereas D33 cows initiated Resynch only when diagnosed not pregnant 33 d after TAI. Cows were classified based on the presence or absence of a corpus luteum (CL) at the not-pregnant diagnosis, and cows without a CL received an intravaginal progesterone-releasing insert during Resynch. When analyzed as a systematic strategy, pregnancy rate per AI (PR/AI) was greater for cows assigned to the D33 than the D26 Resynch treatment (39.4 vs. 28.6%). A treatment × parity interaction was detected for PR/AI after Resynch for nonpregnant cows having a CL in which primiparous cows had a greater PR/AI than multiparous cows when Resynch was initiated 33 d after the initial TAI, and primiparous and multiparous cows when Resynch was initiated 26 d after the initial TAI. Pregnancy loss for Resynch was 6.4% between 33 and 40 d, and 2.6% between 40 and 61 d after Resynch TAI. We concluded that delaying initiation of Resynch until 33 d after TAI increased PR/AI for primiparous cows.     

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.     

A pregnancy detection assay using milk samples: Evaluation and considerations

Two experiments were conducted to evaluate a pregnancy-detection assay based on the measurement of pregnancy-associated glycoproteins (PAG) in milk samples. In experiment 1, milk samples were collected on the day of first pregnancy check (33–52 d postinsemination; n = 119) or second check (60–74 d postinsemination; n = 60). The accuracy in identification of pregnant and nonpregnant cows was 99% at first check. Only 6% of samples were found to be within an intermediate range of PAG concentrations and classified as requiring recheck by the assay. At second check, the accuracy of the assay was 98%. Fifteen percent of these samples were classified as requiring recheck. In experiments 2a (n = 17 cows) and 2b (n = 16 cows), milk and plasma samples were collected from cows at weekly intervals beginning 2 (experiment 2a) or 4 d (experiment 2b) after insemination. The earliest time point at which pregnant cows were accurately classified as pregnant by the assay was on d 30 postinsemination. A transient decline in PAG levels into the intermediate range was observed on d 46 to 72 postinsemination. This coincides with the time of recheck in experiment 1. Results obtained with the plasma samples were essentially the same. The accuracy of pregnancy identification based on milk samples from nonpregnant and pregnant cows was 99%. Levels of PAG in milk were useful in identifying 6 incidences of embryonic mortality. No consistent relationship was noted between the timing of the decline in PAG levels and the timing of luteal regression in this small number of cows.     

Effect of pretreatment with prostaglandin F2alpha before resynchronization of ovulation on fertility of lactating dairy cows

Our objective was to assess the effect of pretreatment with PGF_2α 12 d before initiation of a protocol for resynchronization of ovulation (Resynch) using an Ovsynch protocol. Lactating Holstein cows diagnosed not pregnant 31 d after a timed artificial insemination (TAI) were randomly assigned to initiate the Resynch protocol 32 d after TAI (n = 255; RES), or receive 25 mg of PGF_2α 34 d after TAI and initiate the Resynch protocol 12 d later at 46 d after TAI (n = 272; PGF+RES). Within each treatment, a subset of cows were examined using transrectal ultrasonography to determine ovulatory response to the first GnRH injection of the Resynch protocols or a blood sample was collected to determine serum progesterone (P₄) at initiation of the Resynch protocol, or both. Overall, PGF+RES cows had more pregnancies per artificial insemination (P/AI) than RES cows 66 d after TAI (35.2 vs. 25.6%), whereas pregnancy loss from 31 to 66 d after TAI was greater for RES than PGF+RES cows (17.1 vs. 7.6%). Although P/AI was greater for cows with high (≥1.0 ng/mL) vs. low (<1.0 ng/mL) P₄ at the first GnRH injection of the Resynch protocols, treatment did not affect the proportion of cows with low P₄ at the first GnRH injection of the Resynch protocols. Overall, no effect of treatment on ovulatory response to the first GnRH injection of the Resynch protocols was detected. We conclude that pretreatment with PGF_2α 12 d before initiation of the Resynch protocol increased P/AI 66 d after TAI for cows with serum P₄ concentration >1.0 ng/mL at the first GnRH injection of the Resynch protocol and decreased pregnancy loss from 31 to 66 d after TAI. This modified resynchronization protocol may be a useful strategy for reproductive management of lactating dairy cows.     

Time to increase in pregnancy-specific protein B following artificial insemination is a direct determinant of subsequent pregnancy loss in lactating dairy cows

Increasing progesterone (P4) during early conceptus development may be crucial for establishment of pregnancy in dairy cattle. The objective of this study was to determine if human chorionic gonadotropin (hCG) at various times after ovulation will increase serum P4 during elongation and increase the chances for, and reduce variability to, initial increase in pregnancy-specific protein B (PSPB) following artificial insemination (AI). Time to PSPB increase was defined as the first day of increase in concentrations of PSPB between d 18 and 28 after ovulation in cows with ≥12.5% increases for 3 consecutive days compared with baseline. Lactating cows (n = 368) synchronized to Double-Ovsynch (first service) or Ovsynch (second or greater service) received one of 4 treatments: no hCG (control), or 3,000 IU of hCG on d 2 (D2), 2 and 5 (D2+5), or 5 (D5) after ovulation. All cows were examined via ultrasound on d 5 and 10 postovulation to determine percentage of cows with hCG-induced accessory CL (aCL) and to quantify and measure all luteal structures. Samples for serum P4 were collected on d 0, 5, 19, and 20 postovulation. The P4 was increased in D2, D2+5, and D5 groups compared with control. The D2+5 and D5 treatments increased aCL and P4 compared with D2 and control. The D2 treatment increased P4 on d 5 after ovulation compared with control. Serum PSPB samples were collected daily from all cows on d 18 through 28 after ovulation for determination of d of PSPB increase. Pregnancy diagnoses were performed via ultrasound examination on d 35, 63, and 100 after ovulation and AI. The D5 treatment reduced percentage of cows with, and increased the time to, PSPB increase. Primiparous cows with ipsilateral aCL had reduced pregnancy loss before d 100 postovulation compared with cows with contralateral aCL. Cows that had PSPB increase >21 d postovulation had 4× greater chances of pregnancy loss compared with cows that had PSPB increase on d 20 or 21. The highest quartile of P4 on d 5, but not on d 19 and 20, was associated with reduced time to PSPB increase. Time to PSPB increase appears to be an important measurement to understand reasons for pregnancy loss in lactating dairy cows. Increasing P4 utilizing hCG after ovulation did not enhance early pregnancy or reduce pregnancy losses in lactating dairy cows.     

Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian, conceptus, and insulin-like growth factor system responses

Nonlactating dairy cows were used to examine effects of bovine somatotropin (bST) on components of the insulin-like growth factor (IGF) system. Estrus was synchronized in cows with a Presynch + Ovsynch protocol and timed AI (TAI; n = 55) or not TAI (cycling, C; n = 23) on d 0 (time of synchronized ovulation). On d 0 and 11, cows received bST (500 mg) or no bST, and were sacrificed on d 17. Pregnancy rates were less in bST cows (27.2%, 9 of 33) than in controls (63.6%; 14 of 22). In contrast, conceptuses were larger in bST-treated cows (39.2 +/- 4.8 cm) than in controls (20 +/- 4.3 cm). Total interferon-tau in uterine luminal flushings (ULF) was greater in bST-treated cows (7.15 > 2.36 microg). Number of class 2 follicles (6 to 9 mm) was less in bST-C cows on d 7 and 16. On d 17, corpus luteum (CL) weight tended to be greater in bST-treated cows. Concentrations of progesterone were greater after d 10 in C than in pregnant (P) cows. In the ULF, IGF-binding protein-3 was greater in bST-P cows than in pregnant cows. A tendency for an increase in IGF-I hormone concentrations in the ULF was detected on d 17 in bST-treated and cyclic cows. Endometrial mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 increased in bST-C, but not in bST-P cows. Treatment with bST increased plasma concentrations of insulin, IGF-I, and growth hormone (GH). In conclusion, bST may have hyperstimulated plasma IGF-I and insulin to cause asynchrony between conceptus and uterus that was detrimental to pregnancy.     

Evaluation of two hormonal protocols for synchronization of ovulation and timed artificial insemination in dairy cows managed in grazing-based dairies

To evaluate the efficacy of two hormonal protocols for synchronization of ovulation and timed artificial insemination (TAI) in dairy cows managed in grazing-based dairies, lactating dairy cows (n = 142) from two grazing-based dairies were randomly assigned to one of three treatment groups. Cows in the first group (Ovsynch) received 50 microg of GnRH (d -10); 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) followed by timed AI on d 0. Cows in the second group (PGF + Ovsynch) received a modified Ovsynch and timed AI similar to Ovsynch but with the addition of 25 mg of PGF2alpha 12 d (d -22) before initiation of Ovsynch. Cows in the third group (control) received standard reproductive management in place on each farm. Luteolysis occurred in 90.5% of cows exhibiting luteal function on d -22 in the PGF + Ovsynch treatment group, whereas none of the cows in the Ovsynch group underwent luteolysis on d -22. Synchronization rate (i.e., ovulatory response at 48 h after the second GnRH injection), conception rates at TAI and pregnancy rates after 35 d of breeding were similar for cows in the Ovsynch and PGF + Ovsynch groups. The proportion of anovular cows at the first GnRH injection of the synchronization protocols (d -10) was similar for cows receiving Ovsynch (28.0%) and PGF + Ovsynch (30.7%), and conception rate at TAI was similar for cycling (45.8%) and anovular (30.0%) cows. The cumulative pregnancy rate was greater for cows receiving TAI compared with control cows after 7 d of breeding (41.2 vs. 20.0%) but did not differ at 35 d of breeding (54.9 vs. 60.0%). Administration of PGF2alpha 12 d before initiation of Ovsynch did not improve synchronization, conception, or pregnancy rate compared with the standard Ovsynch protocol. Synchronization of ovulation to initiate timed AI at the onset of the breeding season resulted in earlier establishment of pregnancy compared with standard reproductive management.     

Endogenous and exogenous progesterone influence body temperature in dairy cows

Three experiments were conducted to determine the effect of endogenous progesterone (P4) on body temperature comparing lactating, pregnant with lactating, nonpregnant cows, and to study the effect of exogenous P4 administered via a controlled internal drug release (CIDR) insert on body temperature in lactating dairy cows. Body temperature was measured vaginally and rectally using temperature loggers and a digital thermometer, respectively. In experiment 1, 10 cyclic lactating cows (3 primiparous, 7 multiparous) and 10 lactating, pregnant cows (3 primiparous, 7 multiparous) were included. Vaginal temperatures and serum P4 concentrations were greater in pregnant cows (vaginal: 0.3±0.01°C; P4: 5.5±0.4 ng/mL) compared with nonpregnant cows. In experiment 2, estrous cycles of 14 postpartum healthy, cyclic, lactating cows (10 primiparous, 4 multiparous) were synchronized, and cows were assigned randomly to 1 of 2 treatments (CIDR-P4 or CIDR-blank). A temperature logger was inserted 1 d after ovulation using a P4-free CIDR (CIDR-blank) and a CIDR containing 1.38g of P4 (CIDR-P4) in the control (n=7) and the P4-treated group (n=7), respectively. On d 3 after P4 treatment, vaginal temperature was 0.3±0.03°C greater compared with that on d 1 and d 5. In experiment 3, 9 cyclic multiparous lactating cows were enrolled 1±1 d after confirmed ovulation and a temperature logger inserted. Two days later, a CIDR-P4 was inserted on top of the CIDR-blank. On d 5±1 and d 7±1, respectively, the CIDR-P4 and CIDR-blank with the temperature logger were removed. During the CIDR-P4 treatment (48h), vaginal temperature was 0.2±0.05°C and 0.1±0.05°C greater than during the pre- and post-treatment periods (48h), respectively. Serum P4 concentration peaked during CIDR-P4 treatment (2.2±0.8 ng/mL) and was greater than during the pre-treatment period (0.2±0.2 ng/mL) for 48h. An increase in vaginal temperature could be due to endogenous and exogenous P4. However, a correlation between serum P4 concentrations and body temperature did not exist. Further investigations are warranted to better understand the pathways of the thermogenic effect of P4 on body temperature.