ABSTRACT
The hypothesis that CSF2 plays a role in the preimplantation development of the bovine embryo was tested by evaluating consequences of inactivation of CSF2RA (the functional receptor in the embryo) for development of embryos in utero. CRISPR/Cas9 was used to alter sequences on exon 5 and intron 5 of CSF2RA, Control embryos were injected with Cas9 mRNA only. Embryos > 16 cells at day 5 after insemination were transferred to synchronized recipient females in groups of 7 to 24. Embryos were flushed from the uterus two days later. The proportion of recovered embryos that developed to the blastocyst stage was lower for knockout embryos (39%) than for control embryos (63%). RNA sequencing of individual morulae and blastocysts indicated a total of 27 (morula) or 15 (blastocyst) differentially-expressed genes (false discovery rate <0.05). Gene set enrichment analysis indicated that the knockout affected genes playing roles in several functions including cell signaling and glycosylation. It was concluded that signaling through CSF2RA is not obligatory for development of the bovine preimplantation embryo to the blastocyst stage but that CSF2 signaling does enhance the likelihood that the embryo can become a blastocyst and result in specific changes in gene expression.
ABSTRACT
To determine effects of dam parity on perinatal nutrient availability in beef cattle, data and samples were collected from 18 primiparous and 35 multiparous spring-calving Sim-Angus dams and their calves. Time to stand was recorded and neonatal vigor assessed. Jugular blood was collected from a subset of calves at 0 (post-standing and pre-suckling) 6, 12, 24, 48, and 72 h of age, and blood chemistry panels were completed. Expelled placentas were dissected, dried, and weighed. Prepartum maternal circulating glucose, non-esterified fatty acids (NEFA), triglycerides, and urea N were analyzed. All statistical models included the fixed effect of dam parity, and calf sex (when P ≤ 0.25) was included for calf and placental variables. Effects of sampling hour, and parity × hour were included for calf metabolites over time using repeated measures. Multiparous dams had greater body weight prepartum (P < 0.001) but similar (P = 0.25) body condition score. Maternal circulating urea N and triglycerides were greater (P ≤ 0.05) in multiparous dams pre-calving. Calves born to primiparous dams weighed 10% less (P ≤ 0.04) at birth with smaller (P ≤ 0.01) heart and abdominal girths. Cotyledonary, intercotyledonary, and total placental masses were less (P ≤ 0.05) for primiparous dams. Dam parity did not affect (P ≥ 0.58) calf time to stand, vigor score at 10 min, or rectal temperature. Serum glucose was greater (P = 0.03) at 0 h but less (P ≤ 0.04) at all other hours in calves from primiparous dams. Calves from primiparous dams had greater (P ≤ 0.02) serum NEFA at 6, 12, and 24 h although plasma triglycerides were greater (P < 0.001) at 6 h. Calves from primiparous dams had greater (P ≤ 0.04) serum urea N at 12 h and creatinine at 12 and 24 h. Plasma insulin was greater (P ≤ 0.04) in calves from multiparous dams at 12, 48, and 72 h, but parity did not affect (P ≥ 0.18) serum total protein or plasma cortisol. Serum aspartate aminotransferase was greater (P ≤ 0.04) at 6 and 24 h, creatine kinase was greater at 24 h, and gamma-glutamyl transpeptidase was less (P ≤ 0.04) at 6, 12, and 24 h, for calves from primiparous dams. Calves born to primiparous dams had greater (P ≤ 0.02) total bilirubin and direct bilirubin at 12 and 24 h. Data indicate that calves born to first-parity heifers had decreased perinatal nutrient availability, resulting in reduced fetal and placental growth, as well as greater energy reserve mobilization and metabolic indicators of stress as neonates.
Approximately two-thirds of beef calf deaths prior to weaning occur within the first 3 wk after birth. The goal to have heifers produce their first calf by 2 yr of age likely contributes to factors that limit nutrients available for fetuses and calves immediately after birth. However, little is known about differences in heifers (first parity) and cows (later parities) regarding factors affecting calf resilience, such as fetal growth and calf metabolism shortly after birth. Our data show that calves born to first-parity heifers had altered nutrient availability, demonstrated through smaller placentas, lower birth weights, and altered metabolites in early life. Although calves had similar vigor and ability to maintain body temperature, calves born to first-parity heifers had to mobilize more energy and had lower insulin during the first 3 d post-birth. Calves born to first-parity heifers had greater indicators of stress during the first 72 h of life not associated with calving difficulties. Overall, these effects may have increased morbidity and mortality of calves born to first-parity heifers if they were in a less intensively-managed system. Better understanding of challenges faced by calves born to first-parity dams provides opportunities for their improved management.
Subject(s)
Fatty Acids, Nonesterified , Placenta , Pregnancy , Cattle , Animals , Female , Parity , Fetal Development , Triglycerides , GlucoseABSTRACT
In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.
