Animals
The animal experiment was approved by the Institutional Animal Care and Use Committee of the Gyeongsangbuk-do Livestock Research Institute (Gyeongsangbuk-do, Korea) and all applicable national laws and policies regarding the care and use of animals were observed during the experiment. This study measured hormone levels in 14 cows (51.2 ± 3.8 months, 2.2 ± 0.2 parities, 401.4 ± 10.2 kg). During the experiment, the surrogate mothers were housed in a stanchion barn with sufficient space and were given feed according to the Korean feeding standards. Rice straw, mineral blocks, and water were available ad libitum. At the beginning of the experiment, the cows had a mean body condition score of approximately 2.3 ± 0.03 on the Korea Animal Improvement Association scale of 1 to 5. Animals were excluded if they had abnormalities in the ovaries and uterus detected by transrectal ultrasonography.
Artificial insemination
The AI program was based on an ovulation synchronization protocol [18]. Cows in the AI group (n = 5) were treated with 0.021 mg intramuscular gonadotropin-releasing hormone on days 0 and 9, and 25 mg intramuscular prostaglandin F2α on day 7; they were inseminated 18 h after the injection on day 9.
Oocyte in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of embryos
Ovaries were obtained from a local abattoir and maintained in saline at 35°C during transport to the laboratory. Cumulus-oocyte complexes (COCs) from follicles 2–8 mm in diameter were aspirated using an 18-guage needle. The COCs, those surrounded by more than three layers of cumulus cells and evenly distributed the cytoplasm were sorted. For IVM, COCs were cultured for 22 h in 450 μL TCM-199 supplemented with 0.005 AU/mL follicle-stimulating hormone (F2293; Sigma-Aldrich, USA), 10% fetal bovine serum (GIB16000-044; Thermo Fisher Scientific, USA), 1 μg/mL 17𝛽-estradiol (E4389; Sigma-Aldrich, USA), and 100 μM cysteamine (M6500; Sigma-Aldrich, USA) in a humidified atmosphere of 5% CO2 at 38.5°C.
Motile spermatozoa were refined by the Percoll gradient method [19]. The spermatozoa were purified from thawed semen straws by density-gradient centrifugation on a Percoll discontinuous gradient (45–90%) at 1500 rpm for 15 min. The Percoll density gradient was prepared by layering 1 mL of 45% Percoll solution onto 1 mL of 90% Percoll solution in a 15-mL conical tube. The thawed semen was layered onto the top of the Percoll gradient solution and the tube was centrifuged. The pellet was washed twice with capacitation-Tyrode's albumin lactate pyruvate (TALP) via centrifugation for 5 min at 1500 rpm. The active, motile spermatozoa from the pellet were added to droplets containing matured oocytes. Oocytes were inseminated on day 0 with 1–2 × 106 spermatozoa/mL for 18 h in IVF-TALP medium (NO-100; Nutricell, Brazil) under mineral oil in a humidified atmosphere of 5% CO2 at 38.5°C. The fertilized oocytes were denuded and cultured in two-step chemically defined culture medium (5 days in early stage medium and then 2 days in later stage medium) at 38.5°C in an atmosphere of 5% O2, 5% CO2, and 90% N2 [20].
Embryo transfer and pregnancy diagnosis
A single transgenic bovine embryo was loaded into the central drop of BioLife Transfer & Holding medium (C15C; Agtech, USA) in a 0.25-mL straw with two microdrops side by side, and then sealed. Embryo-loaded straws were transported to Gyeongsangbuk-do Livestock Research Institute in an embryo transporter (TE 100 Compact; WTA Reproduction Technologies, Brazil). One straw was loaded onto a 0.25-mL ET Gun (16301; WTA Reproduction Technologies, Brazil) with minimal contamination. The loaded embryo was transferred to the uterine horn of a recipient by the transcervical method on day 7 (estrus = day 0 = day of fusion) using a non-surgical approach [21]. Surrogates were examined by rectal palpation and ultrasonography 50 days post-estrus to assess embryo survival and pregnancy. Pregnant cattle were checked by rectal palpation and ultrasonography at regular intervals thereafter.
Blood sampling
Blood samples were collected daily beginning 6 days before expected parturition until 7 days after parturition. The samples were collected via jugular venipuncture and stored in EDTA-containing (18.0 mg) collection tubes (Vacutainer 10 mL; Becton Dickinson, New Zealand). After collection, the samples were immediately placed on ice and then transferred to 4°C and stored for 24 h before the plasma was isolated via centrifugation at 1900 × g for 30 min at 4°C. The plasma was stored frozen at −80°C until further analysis.
Enzyme-linked immunosorbent assay (ELISA)
Progesterone, prolactin, and cortisol concentrations were measured using sandwich ELISA assays with the Bovine Progesterone ELISA Kit (NBP2-60122-1; Novus Biologicals, USA), Bovine Prolactin ELISA Kit (OKCD06890; Aviva Systems Biology, USA), and Cortisol Parameter Assay Kit (KGE008B; R&D Systems, USA), respectively, according to the manufacturers’ instructions. For ELISA measurement, progesterone, prolactin, and cortisol were diluted 1, 2, and 30 times, respectively. The sample recovery rate was 80–120% in all tests. Signals were obtained using a SpectraMax 190 microplate reader (Molecular Devices, USA). Hormone levels were quantified by extrapolating the signal into the linear range of a standard curve. SoftMax Pro 7.0.2 (Molecular Devices, USA) was used for data analysis.
Statistical analysis
All results are presented as the mean ± standard error of the mean. Statistical significance was estimated using analysis of variance, followed by Tukey’s multiple correction if not stated otherwise. All statistical analyses were performed using GraphPad Prism 8 (ver. 8.3.0; GraphPad Software, USA).