Exacerbated conceptus signaling does not favor establishment of pregnancy in beef cattle.

BACKGROUND: Insufficient production of anti-luteolytic signals by the pre-attachment embryo is considered a major cause of pregnancy failure in cattle. We tested the hypothesis that transfer of multiple blastocysts (n = 5/recipient) and progesterone (P4) supplementation amplify anti-luteolytic signaling and reduce embryonic losses in beef cattle. Cows detected in estrus (D0; n = 104) were assigned randomly to receive 150 mg of injectable long-acting P4 (iP4) or vehicle (non-iP4) on D4 and transcervical transfer of none or five, grade 1, not-frozen, in vitro-produced blastocysts, on D7. Luteal development and time of structural luteolysis were monitored by ultrasonography. Plasma P4 concentrations were determined on D4, D5 and D7, and daily between D14 and D20. Conceptus signaling was monitored by transcript abundance of interferon-stimulated gene 15 (ISG15) in peripheral blood mononuclear cells isolated on D14, D16, D18 and D20. Early embryonic mortality (EEM) was defined as the absence of ISG15 mRNA upregulation over time and/or luteal regression up to D20. Late embryonic mortality (LEM) was defined as the absence of a conceptus with a heartbeat on pregnancy diagnosis at D30 (PD30) after observing upregulation of ISG15 mRNA and extension of luteal lifespan. Pregnant cows presented conceptuses with heartbeat at PD30. RESULTS: On D5, iP4-treated cows had P4 concentrations 2.07-fold greater than non-iP4 treated (P < 0.001). On D7, P4 concentrations were similar. Pregnant and LEM animals showed a progressive increase in the abundance of ISG15 from D14 to D20. iP4-treated cows detected pregnant at PD30 had 1.53-fold greater abundance of ISG15 mRNA between D14 and D20 than non-iP4 treated cows (P = 0.05). iP4 doubled the frequency of EEM while it did not affect LEM. At PD30, embryonic survival was 37.0% vs. 55.6% for iP4-treated vs. control cows. Majority of pregnant cows (71%) presented only a single viable embryo. CONCLUSIONS: A substantial proportion of cows had EEM (31%) and LEM (20%) even after transferring multiple blastocysts. This argues that mortality was due to poor uterine receptivity that could not be reversed by supplemental P4 or overcome by transferring multiple blastocysts. Further, a given uterine environment was not necessarily adequate to all embryos.

Molecular characteristics of granulosa and cumulus cells and oocyte competence in Nelore cows with low and high numbers of antral follicles.

The aims of this study were to investigate whether the number of antral follicles (AF) in the ovaries of Nelore cows is influenced with the developmental competence of oocytes to reach the blastocyst stage and to quantify the mRNA abundance of genes associated with folliculogenesis and oogenesis in granulosa and cumulus cells. A total of 168 cows were distributed into two experimental groups according to the number of AF, low (≤31) and high AF (≥92), which were determined based on the mean number of AF (61.14) ± SD (30.43). Granulosa and cumulus cells were used to assess the mRNA expression of 16 genes. Cumulus cells from cows with low AF had higher mRNA expression of genes involved in meiosis resumption (NPR-2, NPR-3) and cumulus cell expansion (FGF10), as well as a transcription factor involved in the regulation of oocyte maturation and cell proliferation (STAT3). Conversely, granulosa cells from females with high AF had higher expression of PGR and AMHR2a, which are involved in meiosis resumption and cumulus cell expansion. Cumulus-oocyte complexes (COCs) were collected from 356 cows with low and high AF populations to evaluate embryo development. Cleavage and blastocyst rates did not differ between the groups. In conclusion, our findings revealed that genes involved in folliculogenesis and oogenesis are differently expressed in cumulus and granulosa cells of cows having low and high numbers of AF. These molecular differences suggest that the regulation of oocyte maturation, meiotic resumption and cumulus expansion may be influenced by the number of AFs. However, the variations in gene expression were not associated with in vitro oocyte developmental competence to reach the blastocyst stage, which confirms that oocytes from Nelore cows with low and high numbers of AF are similarly able to mature, regulate the fertilization process and support pre-implantation embryo development.

Pregnancy Rates to Fixed Embryo Transfer of Vitrified IVP Bos indicus, Bos taurus or Bos indicus × Bos taurus Embryos.

The pregnancy rates obtained after the transfer of cryopreserved in vitro-produced (IVP) embryos are usually low and/or inconsistent. The objective of this study was to evaluate the pregnancy rates of Holstein, Gyr and Holstein × Gyr cattle after the transfer of vitrified IVP embryos produced with X-sorted sperm. Seventy-two Gyr and 703 Holstein females were subjected to ovum pickup (OPU) sessions, followed by in vitro embryo production using semen from sires of the same breeds. Embryos (1636 Holstein, 241 Gyr and 1515 Holstein × Gyr) were exposed to forskolin for 48 h prior to vitrification. The pregnancy rate achieved with Gyr dam and sire was 46.1%, which was similar (p = 0.11) to that of Holstein dam and Gyr sire (40.3%). Crossing Gyr dams with Holstein sires resulted in a pregnancy rate of 38.9% and did not differ (p = 0.58) from the pregnancy rate obtained with the cross between Holstein dams and Gyr sires. The rate obtained with Holstein dam and sire was 32.5%. The average pregnancy rate was 36.6%, and no difference was found in the proportion of female foetuses (88.8%, in average) among breeds (p > 0.05). In conclusion, transfer of cryopreserved X-sorted embryos represents an interesting choice for dairy cattle. Despite the small differences between pregnancy rates, we highlight the efficiency of this strategy for all of the racial groups studied.

Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle.

Obtaining sexed sperm from previously frozen doses (reverse-sorted semen [RSS]) provides an important advantage because of the possibility of using the semen of bulls with desired genetic attributes that have died or have become infertile but from whom frozen semen is available. We report the efficiency of RSS on the pregnancy rate and birth rate of calves in a large-scale program using ovum pick-up and in vitro embryo production (IVEP) from Bos indicus, Bos indicus-taurus, and Bos taurus cattle. From 645 ovum pick-up procedures (Holstein, Gir, and Nelore), 9438 viable oocytes were recovered. A dose of frozen semen (Holstein, Nelore, Brahman, Gir, and Braford) was thawed, and the sperm were sex-sorted and cooled for use in IVF. Additionally, IVF with sperm from three Holstein bulls with freeze-thawed, sex-sorted (RSS) or sex-sorted, freeze-thawed (control) was tested. A total of 2729 embryos were produced, exhibiting a mean blastocyst rate of 29%. Heifers and cows selected for adequate body condition, estrus, and health received 2404 embryos, and 60 days later, a 41% average pregnancy rate was observed. A total of 966 calves were born, and 910 were of a predetermined sex, with an average of 94% accuracy in determining the sex. Despite the lower blastocyst rate with freeze-thawed, sex-sorted semen compared with sex-sorted semen, (P < 0.05), the pregnancy rate (bull I, 45% vs. 40%; II, 35% vs. 50%; and III, 47% vs. 48% for RSS and control, respectively; P > 0.05) and sex-sorted efficiency (bull I, 93% vs. 98%; II, 96% vs. 94%; and III, 96% vs. 97% for RSS and control, respectively; P > 0.05) were similar for each of the three bulls regardless of the sperm type used in the IVF. The sexing of previously frozen semen, associated with IVEP, produces viable embryos with a pregnancy rate of up to 40%, and calves of the desired sex are born even if the paternal bull has acquired some infertility, died, or is located a long distance from the sexing laboratory. Furthermore, these data show the feasibility of the process even when used in a large-scale IVEP program.

Cryosurvival and pregnancy rates after exposure of IVF-derived Bos indicus embryos to forskolin before vitrification.

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 µM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates.

Comparison of synthetic oviductal fluid and G1/G2 medium under low-1 oxygen atmosphere on embryo production and pregnancy rates in Nelore (Bos indicus) cattle.

In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low-oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low-oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.