Hormonal estrus synchronization followed by heat detection of recipient animals is a widely used technique in embryo transfer (ET) programs around the world. However, the successful outcomes of ET rely heavily on appropriate heat detection of recipient animals. Additionally, limited availability of personnel and time to detect heat makes ET impractical at times. Therefore, pregnancy rates (PR) following a timed embryo transfer (TET) program were evaluated taking into account different variables of recipient cows, donor embryos, and season of transfer under field conditions in Argentina. Recipient cows were synchronized utilizing a progesterone (P4) intravaginal device (DIB, Syntex, Argentina) for 7 days. On Day 0 (day of DIB insertion) 2 mg of estradiol benzoate (EB) i.m. was administered and immediately after DIB removal on Day 7, 500 μg of D(+) Cloprostenol i.m. (PG, Ciclase, Syntex), 400 IU of eCG i.m. (Novormon, Syntex) plus 1 mg of cypionate estradiol i.m. (ECP; König, Agentina) were administered to all recipient cows. Fresh (n = 1746) and frozen (n = 2047) beef bovine embryos were nonsurgically transferred into recipient cows on Day 9 after PG. The effects of body condition score (1–9, BCS I: ≤5, n = 804 and BCS II: ≥6, n = 2989), corpus luteum (CL) score (CL1: prominent papilla, n = 932, CL2: small papilla, n = 2028, and CL3: asymmetric, n = 833), location of embryo placement in uterine horn (upper, n = 3566, and lower, n = 227), and type of recipient such as heifer (n = 487), lactating (n = 1010), or dry cows (n = 2296) on PR were evaluated. Additionally, stage of embryo development (4–7), quality (1–3), and season of transfer (spring, summer, fall, and winter) were included in the analysis. Pregnancy rate was confirmed by rectal palpation 60 days after ET. Data were first screened by chi square and then analyzed by Proc Logistic of SAS (SAS Institute Inc., Cary, NC). Overall PR was 54.7%. body condition score, CL, type of recipients, stage of embryo development, and year were not significantly (P > 0.05) associated with PR. As expected, cows that received direct fresh embryos had 1.49 times greater odds of conceiving (odds ratio, OR = 1.49, 95% CI: 1.22 to 1.82) than those receiving frozen embryos. Additionally, cows receiving embryos graded as quality 1 had greater probability to conceive (OR = 1.62, 95% CI: 1.34 to 1.95, or OR = 1.64, 95% CI: 1.25 to 2.21) compared to quality 2 or 3 respectively. Moreover, embryos placed into the upper portion of uterine horn were more likely (OR = 1.48) to conceive than those placed in the lower uterine portion. Regardless of embryos and recipient cow variables analyzed, there was a greater probability for cows to conceive during winter (OR = 1.41, 95% CI: 1.13 to 1.95) as compared with summer. In conclusion, synchronized recipient animals for a TET program resulted in acceptable PR for both fresh and frozen embryos. Results suggested that the quality and location of embryos (upper portion) in uterine horn were the most important variables associated with PR in recipient cows. Therefore, TET is a viable and practical option allowing a successful outcome for many beef operations where availability of personnel is limited.

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In a large-scale bovine cloning program intended to obtain transgenic animals, it is important to maximize the number of calves produced. The present experiment was designed to test the hypothesis that different transfection events of the same somatic cell line can affect embryo and/or fetal survival. A fetal cell line was established from a 75-day-old Jersey female fetus. It was used as control and was also transfected 3 times with the same protocol. They were named Transfection 1, 2, and 3. Genetically modified cells were produced and isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL-HEPES with 1 mg mL−1 bovine testis hyaluronidase. Metaphases were assessed, and oocytes were enucleated by visualization with Hoechst 33342 (5 µg mL−1) under UV light (<6 s). Donor cells from different treatments were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium with an atmosphere of 5% CO2 + 5% O2 + 90% N2. Development to blastocysts (Days 7 to 9) was recorded. Two blastocysts were transferred nonsurgically per recipient cow, and pregnancies at 30 days were determined by ultrasonography. All data were analyzed by chi-square test. In vitro development to blastocysts was similar in all treatment groups. One birth was obtained from the control. Four and 7 births were obtained from Transfections 1 and 3, respectively. Although Transfection 2 had good in vitro development, this treatment did not produce any pregnancy. This fact demonstrated that the transfection event provides an additional source of variability in obtaining live transgenic animals. Our results pointed out the necessity to monitor fetal survival by ultrasonography in order to detect as soon as possible any deficiencies in development introduced by transfection. Table 1.Effect of different transfection events of same line on embryo and fetal survival

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