Optimizing ReBreed21 I: Evaluation of endocrine and ovarian dynamics in non-bred Bos indicus heifers.

The present study evaluated follicular and endocrine dynamics during ReBreed21, a reproductive strategy that allows resynchronization of ovulation every 21 days in Bos indicus (Nelore) heifers. A synchronized estrous cycle was induced using a standard timed ovulation protocol (d -10: P4 implant inserted + 2 mg estradiol benzoate; d -2: P4 removed+ 0.5 mg cloprostenol + 0.6 mg estradiol cypionate + 200 IU equine chorionic gonadotropin (eCG); d0: 8.4 µg buserelin) without AI to ensure nonpregnancy in heifers. Day of GnRH was designated d0 of estrous cycle. On d12, heifers (n = 80) were randomized into three experimental groups: (1) ReBreed21 (n = 28) d12 P4 device inserted, d19 P4 device withdrawal plus 200 IU eCG, and d21 8.4 µg buserelin (GnRH); (2) ReBreed21+G (n = 26) same as ReBreed21 plus GnRH (16.8 µg) treatment on d12; and (3) Control (n = 26) no treatment. ReBreed21+G increased two-fold (62.9%; 18/26) percentage of heifers with synchronized follicular wave emergence compared to Control (34.6%; 9/26) whereas ReBreed21 (53.6%; 15/28) was intermediate. The ReBreeed21 groups (eCG on d19) increased (P < 0.01) follicular growth between d19 and d21 in ReBreed21 (2.3 ± 0.2 mm) and ReBreed21+G (3.4 ± 0.2 mm) compared with Control (1.2 ± 0.3 mm), resulting in greater (P < 0.01) follicle diameter on d21 for ReBreed21 (10.7 ± 0.4 mm) and ReBreed21+G (10.8 ± 0.4 mm) compared with Control (9.1 ± 0.5 mm). Structural luteolysis was similar among groups (P = 0.51), although the average day when P4 was <1 ng/mL was later (P < 0.01) for ReBreed21 (20.5 ± 0.2) and ReBreed21+G (20.7 ± 0.2) compared to Control (19.2 ± 0.4). Overall ovulation at the end of the estrous cycle was increased (P = 0.03) for ReBreed21 groups (83.3%; 45/54) compared with Control (57.7%; 15/26). Synchronized ovulation on day 22-23 was greater (P < 0.01) for ReBreed21 (78.6%; 22/28) and ReBreed21+G (76.9%; 20/26) compared with Control (30.8%; 8/26). Thus, the ReBreed21 resynchronization program produced acceptable endocrine and follicular dynamics, including synchronized ovulation at the end of the protocol in nonpregnant heifers providing good rationale for testing the fertility and practical implementation of this protocol under field conditions.

Regional and sectorial impacts of the Covid-19 crisis: Evidence from electronic payments.

We use novel and comprehensive monthly data on electronic payments, by municipality and sector, together with cash withdrawals, to study the impact of Covid-19 in Portugal. Our difference-in-differences event study identifies a causal decrease of 17 and 40 percentage points on the year-on-year growth rate of overall purchases in March and April 2020. We document a stronger impact of the crisis in more central and more urban municipalities, due to a combination of the sectorial composition effect of the local economy and the sharper confinement behavioral effect in these locations. We discuss the importance of tourism for the results.

Development of a novel 21-day reinsemination program, ReBreed21, in Bos indicus heifers.

The aim was to develop a program for resynchronization of ovulation (ReBreed21) that allowed reinsemination of non-pregnant Bos indicus heifers every 21 d using timed AI (TAI) without the need for detection of estrus. The Rebreed21 program begins 12 d after previous TAI (Day 0) by inserting an intravaginal P4 implant (Day 12) that is removed 7 d later (Day 19) combined with treatment with 300 IU of eCG. On Day 21, early pregnancy diagnosis (Doppler PD) is performed based on CL vascularity. Non-pregnant (NP) heifers immediately received AI combined with 100 µg of GnRH. The program is replicated 12 d after second TAI to produce a breeding season (BS) of 42 d with 3 potential TAIs. Two experiments were conducted as a proof of concept for this rapid rebreeding program. In Experiment 1, 76 heifers were enrolled in ReBreed21, as explained above. In Experiment 2, 300 Nellore heifers were synchronized for 1st TAI and randomly assigned to one of two groups: ReBreed21 (n = 147) or another early resynchronization procedure, Resynch14 (n = 153) with P4 implant inserted 14 d after previous TAI plus 50 mg of long-acting injectable P4; 8 d later P4 implant removed (Day 22) and early Doppler PD performed; NP heifers received 150 µg of cloprostenol, 0.5 mg of ECP, and 300 IU of eCG with TAI on Day 24. In both experiments, the largest follicle (LF) was measured at each Resynch TAI. Ultrasound was later used to confirm the early Doppler PD and to determine ovulation (OV) to Resynch at 12 d after TAI in ReBreed21 (Day 33 of pregnancy) and 14 d after TAI in Resynch14 (Day 38 of pregnancy). Final PD was performed 40 d after 3rd TAI. Results for Experiment 1 were: diameter of LF 11.8 ± 0.23 mm; 88.9% OV; 20.5% false positives; 38.1% P/AI at 1st TAI; 44.4% overall P/AI for ReBreed21 TAIs; 72.3% total pregnant at end of BS. In experiment 2, Rebreed21 vs. Resynch14 were different for: diameter of LF (10.9 ± 0.17 vs. 10.0 ± 0.17 mm, P = 0.0003), heifers with LF < 8.5 mm (10.2 vs. 26.4%, P = 0.04), or LF ≥ 11 mm (50.0 vs. 37.2%, P = 0.001), and P/AI at first TAI (29.3% [43/147] vs. 20.3% [31/153], P = 0.074) but similar for OV (overall 86.8% [239/275], P = 0.82), false positives (P = 0.52) overall P/AI for Resynch TAIs (33.6 vs. 28.8%, P = 0.4), and total pregnant at end of BS (58.5% [86/147] vs. 55.6% [85/153], P = 0.64). In addition, median time to pregnancy was 9 d earlier (P = 0.0007) for ReBreed21 than Resynch14. Thus, ReBreed21 is a novel protocol that allows earlier reinseminations than Resynch14 but with similar fertility.

Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF).

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.