Comparison of gonadotropin-releasing hormone and estradiol benzoate plus gonadotropin-releasing hormone to initiate a progesterone-based timed artificial insemination resynchronization protocol in lactating dairy cows.

The present study compared 2 strategies to initiate a progesterone (P4)-based timed artificial insemination (TAI) protocol for lactating dairy cows: only GnRH or estradiol benzoate (EB) plus GnRH (EB+GnRH). Lactating Holstein cows (n = 487; 184 primiparous and 303 multiparous) from 2 commercial dairy herds were used for their second or greater services postpartum. Each week, cows that were nonpregnant at the pregnancy diagnosis 32 d after a previous AI were randomly assigned to 1 of 2 experimental groups that differed only in the strategy to initiate (d 0) the TAI protocol. On d 0, every cow received a 2.0-g P4 implant; in the EB+GnRH group, cows were treated with 2.0 mg i.m. of EB and 16.8 µg i.m. of the GnRH analog buserelin acetate, whereas in the GnRH group, cows received only 16.8 µg i.m. of GnRH. On d 7 after the initial treatment, 0.530 mg i.m. of cloprostenol sodium (PGF) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg i.m. of estradiol cypionate and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Only conventional Holstein semen was used throughout the study. The percentage of cows with corpus luteum (CL) on d 0 (73%) and overall ovulation rate after d 0 (54%) did not differ between groups. The CL regression between d 0 and the first PGF treatment was greater in the EB+GnRH group than the GnRH group (42% vs. 31%). Consequently, the proportion of cows with CL at PGF was greater when only GnRH was used on d 0 compared with EB+GnRH (86% vs. 82%), and the mean number of CL at PGF was greater (1.23 vs. 1.11). The expression of estrus near TAI was greater in GnRH group (84% vs. 77%), and cows showing estrus had greater (44% vs. 10%) pregnancy per AI (P/AI) on d 32 for both treatments. We found no effect of the presence of CL on d 0 or at PGF, nor of ovulation after d 0 or CL regression between d 0 and d 7 on fertility. However, fertility was critically impaired when cows did not have CL at both times, d 0 and at PGF treatment. We did not observe any interaction between treatment and other variables, and the P/AI was similar in cows receiving EB+GnRH or only GnRH on d 0 (37.8% vs. 36.6%). In summary, although there was no detectable difference in P/AI between treatments, this study demonstrated potential negative physiological outcomes caused by EB treatment on d 0 (greater incidence of luteolysis after d 0 and fewer cows with CL at PGF treatment). Overall, we found no benefit of adding EB at the initiation of a P4-based TAI protocol on fertility compared with using GnRH alone, despite differences in ovarian dynamics and expression of estrus.

Interactions of circulating estradiol and progesterone on changes in endometrial area and pituitary responsiveness to GnRH†.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.

Reproductive outcomes of prepubertal Bos indicus beef heifers raised in a pasture-based feeding system submitted to ovulation induction strategies prior to a timed-artificial insemination protocol.

Reproductive outcomes were evaluated in Nelore (Bos indicus) heifers submitted to one, two or no ovulation induction protocols based on progesterone (P4) and estradiol (E2) prior to a timed-artificial insemination (TAI) protocol. A total of 1,437 heifers (13.0 ± 0.8 mo old; 3.1 ± 0.1 of body condition score [BCS] and 279.9 ± 25.8 kg of body weight [BW]) were randomly assigned to 1 of 3 treatments: 0IND (n = 486): no ovulation induction protocol; 1IND (n = 481): one ovulation induction protocol; or 2IND (n = 470): two ovulation induction protocols. On Day -47, heifers from 2IND received a disinfected intravaginal P4 device (2 g, previously used for 21 d), kept until Day -40, when 0.5 mg of E2 cypionate (EC) was given. On Day -19, heifers from 2IND and 1IND underwent the same protocol. On Day 0, all heifers were submitted to the same TAI protocol, starting with a P4 device (0.5 g), 0.5 mg of cloprostenol sodium (PGF), and 1.5 mg of E2 benzoate. On Day 7, P4 device was removed, 0.5 mg of PGF, 0.5 mg of EC, and 200 IU of equine chorionic gonadotropin (eCG) were administered. The TAI was performed 2 d later (Day 9). Blood samples were collected on Days -47 and 0, to determine the presence of CL (circulating P4 concentrations ≥ 1.0 ng/mL). Ultrasound was performed on Days 40, 75 and between Day 150 and parturition to assess pregnancy per AI (P/AI) and pregnancy loss (PL). Statistical analyses were performed using SAS 9.4 (a-cP ≤ 0.05; A,B0.05 < P ≤ 0.10). The proportion of heifers with CL on Day -47 was similar among groups (3.4%). A greater proportion of heifers from 1IND had CL on Day 0, followed by 2IND, then 0IND (87.9a; 80.4b; 28.8c%). There was an effect of treatment on expression of estrus (2IND: 66.6a; 1IND: 67.2a; 0IND: 57.4b%), P/AI on Day 40 (2IND: 53.4a; 1IND: 43.9b; 0IND: 46.5b%), P/AI on Day 75 (2IND: 49.8a; 1IND: 40.5b; 0IND: 44.4ab%) and final P/AI (2IND: 45.5a; 1IND: 35.8b; 0IND: 40.5ab%). No differences were observed in PL (40-75 = 6.3%; 75-final = 9.6%; Total = 15.3%). Particularly within lighter heifers, there was an effect of treatment on P/AI on Day 40 (0IND: 39.2b; 1IND: 43.3ab; 2IND: 53.9a%) and on Day 75 (0IND: 36.6B; 1IND: 39.0AB; 2IND: 48.5A%). At the first pregnancy diagnosis, more nonpregnant heifers from 2IND had CL on Day 40 than 0IND, but 1IND did not differ from the other groups (85.4a; 74.8b; 80.8ab%). In conclusion, ovulation induction protocols performed prior to the TAI protocol increased the proportion of heifers with CL on Day 0. The use of two induction protocols resulted in greater fertility, particularly in lighter heifers, and increased cyclicity among nonpregnant heifers. These results indicate that this strategy may be an optimized method for inducing cyclicity and enhancing fertility of prepubertal Nelore heifers raised in pasture-based feeding systems.

Timed-artificial insemination protocols for Bos indicus beef heifers: Evaluation of protocol length and variations in prostaglandin F2α treatments.

The aim was to compare reproductive outcomes of Nelore heifers submitted to timed AI (TAI) protocols, with 7 or 9 d of permanence of the intravaginal progesterone (P4) device and different times of prostaglandin F2α (PGF) administration, for first (n = 935) and second (n = 530) services. On Day -24, heifers without corpus luteum (CL) underwent a protocol for induction of ovulation. On Day 0, heifers received a P4 device (0.5 g) and 1.5 mg estradiol (E2) benzoate. In order for the TAI to be carried out on the same day, these treatments were performed 2 d later on the heifers treated with the 7-d protocol. Additionally, heifers received 0.5 mg PGF at different times, resulting in four experimental groups: 9dP4-PGFd9 (n = 365); 9dP4-PGFd7 (n = 369); 9dP4-PGFd0&9 (n = 364); 7dP4-PGFd0&7 (n = 367). These nomenclatures indicate for how many d the P4 device was kept and the specific day on which PGF was given. At P4 removal, all heifers received 0.5 mg E2 cypionate and 200 IU eCG, and TAI was performed 2 d later. Effects were considered significant when P ≤ 0.05 (superscript letters a,b) whereas a tendency was assumed when 0.05 < P ≤ 0.10. Groups 9dP4-PGFd0&9 and 7dP4-PGFd0&7 had lower percentage of heifers with CL at P4 removal. The diameter (mm) of the dominant follicle (DF) was affected by treatment at P4 removal (9dP4-PGFd9: 11.3 ± 0.3b; 9dP4-PGFd7: 11.8 ± 0.2ab; 9dP4-PGFd0&9: 12.6 ± 0.2a; 7dP4-PGFd0&7: 10.8 ± 0.2c) and at TAI (9dP4-PGFd9: 12.7 ± 0.3ab; 9dP4-PGFd7: 13.2 ± 0.2a; 9dP4-PGFd0&9: 13.4 ± 0.2a; 7dP4-PGFd0&7: 12.4 ± 0.3b). Expression of estrus (%) was affected by treatment (9dP4-PGFd9: 89.6a; 9dP4-PGFd7: 93.5a; 9dP4-PGFd0&9: 88.2ab; 7dP4-PGFd0&7: 85.6b). There were no differences among treatments for P/AI on Day 40 (30-35 d post AI), final P/AI (between Day 70 and parturition) and pregnancy loss (between Day 40 and final P/AI). When the permanence of the P4 device was compared, regardless of PGF treatments, 9-d protocols resulted in greater DF diameter at P4 removal and at TAI, and greater expression of estrus (90.4 vs. 85.6%) than the 7-d protocol. Despite that, the 7-d protocol resulted in greater P/AI on Day 40 (55.3 vs. 49.1%). In addition, there was an interaction between protocol duration and body weight, in which heavier heifers (≥ 307 kg) had greater P/AI when treated with the 7-d protocol, in comparison to 9-d. In conclusion, longer TAI protocols (9 d of P4 device duration) resulted in greater DF diameter and expression of estrus. However, the shorter TAI protocol (7 d of P4 device duration) produced greater P/AI on Day 40, particularly in heavier heifers. Within 9-d protocols, the additional dose of PGF on Day 0 or the anticipation of the PGF to Day 7 did not influence fertility.

Improved fertility following a gonadotropin-releasing hormone treatment on day 2 of an estradiol and progesterone-based timed-artificial insemination protocol in lactating dairy cows.

The present study evaluated the addition of gonadotropin-releasing hormone (GnRH) concomitant or 2 d after the beginning of protocols initiated with estradiol benzoate (EB). A total of 459 multiparous and 371 primiparous lactating Holstein cows were enrolled in the study. Weekly cohorts of cows were randomly assigned to 1 of 3 experimental groups that differed in the strategy to initiate the timed AI (TAI protocol. On d 0, all cows received a 1.55-g progesterone (P4) implant. Additionally, cows in the EBd0 group received 2 mg of EB i.m.; cows in the EBd0-GnRHd0 group were treated simultaneously on d 0 with 2 mg of EB plus 100 µg of gonadorelin diacetate tetrahydrate (GnRH) i.m.; and cows in the EBd0-GnRHd2 group received 2 mg of EB on d 0 and 100 µg of GnRH 48 h later (d 2). The remaining treatments in the protocol were similar among groups and included 0.53 mg (i.m.) of cloprostenol sodium (PGF2α) on d 7, followed by a second PGF2α treatment on d 9 (at the time of P4 implant withdrawal) and 1 mg of estradiol cypionate i.m. Then, TAI was performed on d 11 (48 h after P4 removal) in all experimental groups. We detected an effect of treatment on pregnancy per AI (P/AI) on d 30, in which cows from the EBd0-GnRHd2 group demonstrated greater fertility than EBd0 cows, whereas cows in the EBd0-GnRHd0 group did not differ among EBd0 and EBd0-GnRHd0 (40.5 vs. 30.4 vs. 34.4%, respectively). In summary, GnRH treatment at the beginning of an estradiol and P4-based TAI protocol increased fertility only when GnRH was given on d 2. Moreover, a more pronounced positive effect of this strategy was observed in particular classes of cows: multiparous cows, cows with greater milk production, and those receiving the first service.

Influences of the Seasons of the Year and Physiographic Regions on the Levels of Calcium, Copper and Zinc in the Hoof Capsule of Foals Pre- and Postweaning Raised in Native Pasture.

This study evaluated the levels of calcium (Ca), copper (Cu) and zinc (Zn) in the hoof of Criollo foals, pre- and postweaning, correlating them with the concentrations of these inorganic chemical compounds in soil and pasture in the different seasons of the year. We used 41 preweaning foals and 28 postweaning foals. Seventeen forage samples were collected in four seasons, later 10 soil samples per property in five distinct physiographic regions (Santa Maria, Dilermando de Aguiar, Jaguarão, Arroio Grande and Pelotas). There was variability in Ca, Cu and Zn concentrations in the hoof foal between distinct physiographic regions (P < .05), and between seasons. There was no association of preweaning and postweaning Ca levels with forage in the different physiographic regions. The concentrations of Cu and Zn showed positive association (R = 0.69, 0.43; P < .05) with the native pastures in the different physiographic regions in preweaning. In postweaning, only Cu was positively associated (R = 0.74; P < .05). Different season and physiographic regions influence the levels of Ca, Cu and Zn in the hoof capsule and native pasture during the pre- and postweaning stages.

Hormonal combinations aiming to improve reproductive outcomes of Bos indicus cows submitted to estradiol/progesterone-based timed AI protocols.

The aim was to study reproductive outcomes of Nelore (Bos indicus) cows submitted to a 7-d estradiol (E2)/progesterone (P4)-based timed artificial insemination (TAI) protocol, receiving various combinations of doses and hormones. Primiparous (n = 962) and multiparous (n = 1935) cows were submitted to synchronization (n = 2012) and resynchronization (n = 885 non-pregnant cows at pregnancy diagnosis 30 d after TAI) protocols, following a 2 × 2 × 2 factorial arrangement of eight treatments. At the initiation of the TAI protocol (Day -9), all cows received a 1.0 g intravaginal P4 insert, 2.0 mg E2 benzoate and received (PGF1) or not (PGF0) 0.5 mg cloprostenol sodium (PGF). On Day -2, the P4 insert was removed, all cows received 0.5 mg PGF, 300 IU equine chorionic gonadotropin (eCG) and 0.5 (EC0.5) or 1.0 mg estradiol cypionate (EC1.0). On Day 0, cows were treated (G1) with 8.4 µg buserelin acetate (GnRH) or not (G0), concurrently with TAI. The eight treatments were generated: 1) PGF0-EC0.5-G0 (n = 364), 2) PGF0-EC0.5-G1 (n = 363), 3) PGF1-EC0.5-G0 (n = 363), 4) PGF1-EC0.5-G1 (n = 360), 5) PGF0-EC-1.0-G0 (n = 360), 6) PGF0-EC1.0-G1 (n = 363), 7) PGF1-EC1.0-G0 (n = 361), and 8) PGF1-EC1.0-G1 (n = 363). Pregnancy per AI (P/AI) was greater at first AI compared with resynchronization (58.9 [n = 2012] vs. 54.9% [n = 885]). Presence of CL on Day -9 resulted in more cows expressing estrus (81.3 [n = 680] vs. 67.1% [n = 2033]) and greater P/AI (66.0 [n = 692] vs. 54.9% [n = 2106]). There was no difference in P/AI between cows that received or not PGF on Day -9 (58.7 [n = 1447] vs. 56.6% [n = 1450]). In contrast, PGF tended to increase P/AI of cows with CL on Day -9 (with PGF = 69.1 [n = 375] vs. without PGF = 62.5% [n = 317]). Cows that received 1.0 mg EC expressed more estrus than those treated with 0.5 mg (73.8 [n = 1414] vs. 67.9% [n = 1398]) and had greater P/AI (60.2 [n = 1447] vs. 55.1% [n = 1450]). P/AI was greater in cows treated with GnRH at TAI (59.8 [n = 1449] vs. 55.5% [n = 1448]), particularly in cows that did not show estrus (52.7 [n = 393] vs. 38.1% [n = 420]). Moreover, GnRH on Day 0 increased P/AI in cows with BCS < 3.0 (57.1 [n = 723] vs. 48.6% [n = 698]), in primiparous (50.1 [n = 465] vs. 41.9% [n = 497]) and in cows that received 0.5 mg EC (58.9 [n = 723] vs. 51.3% [n = 727]). In conclusion, 1.0 mg of EC on Day -2 and GnRH at TAI improved P/AI, but the combination of a higher dose of EC and GnRH treatment at AI did not enhance this effect. Furthermore, GnRH improved P/AI especially in Bos indicus cows with lower expression of estrus, such as primiparous, thinner cows, and cows treated with 0.5 mg of EC.