2.1 | Animal allocation and experimental design.
This study was conducted between October 2021 and April 2022. Animal experiments described in this article were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals Mansoura University (VM.R.22.12.36). Non lactating pluriparous Ossimi female sheep (n=20) were investigated during the period of this study. Each having three previous parities. The average age of the experimental ewes was 5.5 ± 2.4 y old (mean ± SD, range 3-10 y). The animals were housed in a barn with access to outside runs during the winter, but they were maintained on pasture during late fall and early winter. The base diet during the latter period included pellet feed (amount adjusted to the stage of pregnancy), hay, and mineral supplementation in addition to unlimited water. The experimental animals were allocated into two temperamental groups more reactive and less reactive (LR) after Arena test, a behavioral test used on all the sheep according to Elmetwally et al. (2021) (Elmetwally et al. 2021).
Sheep in the present experiment were synchronized with prostaglandin F22α [PGF2α], analog (125 µg cloprostenol, Schering Plough, USA, i.m) and then mated with a proven fertile male during behavioral estrus. B mode ultrasonography examination was performed on the 25th-day post-breeding. No fetal loss was detected in this study. Arena tests for experimental sheep allocation were done according to previous literature (Elmetwally et al. 2021).
2.2 | Color Doppler sonography examination of uterine arteries
Elmetwally et al. (2016) described the use of a LOGIQ 5 Pro ultrasound device (General Electrics Healthcare, Solingen) equipped with a linear-array, multifrequency (5-10 MHz) transducer for uterine artery localization and Doppler examination in pregnant sheep (Elmetwally et al. 2016a). In each uterine artery corresponding to the gravid side, TAMV, PI and RI were measured (Figure 1). The Doppler angle was kept between 30 and 40, and the indexes were taken every two weeks from week 2 to week 20 of the pregnancy. The Doppler examination took about 30 minutes for each pregnant sheep. The gestational periods of the experimental sheep that delivered normally and without complications were recorded. The weights of the lambs were taken immediately after birth.
2.3 | B-mode sonography
When possible, the following fetometric endpoints were measured using a 9 MHz linear-array transducer (Sonoscape A5V, Shenzhen, China) from wk 2 after breeding until parturition: diameter of amniotic vesicles (figure 2a: AVD), fetal chest (figure 2b: FCHD), and umbilical cord diameter close to the fetal body (figure 2c: UMD), metacarpal length (figure 2d: MCL) according to Elmetwally, 2012 (Elmetwally 2012).
2.4 | Tissue sampling and Macroscopic examination
Placental tissue specimens will be collected from 5 ewes directly after dropping off the placenta (30 minutes to 21 hours after birth). Macroscopic inspection will be performed on placental samples. Sections of placenta from all sheep will be collected as rapidly as possible and stored in 10% buffered formaldehyde for histological analyses.
Histological analysis
Light microscope tissue fixation and processing
All the collected tissue for light microscopy will be perfusion-fixed with 10% buffered formaldehyde for 72 h, rinsed in phosphate buffered saline (PBS; pH 7.4). placenta from random locations will be trimmed and dehydrated in ascending concentrations of ethyl alcohol, cleared in xylol and impregnated embedded paraffin wax using established methods. Sections of 5μm will be collected on glycerol-albumin-coated glass slides and dried for at least 24 h in a 37˚C incubator. Paraffin sections kept in the incubatorwill be prepared for staining.
Tissue staining
Sections of all collected placental tissues were preserved in 10% buffered formalin according to routine protocols (figure 3). Following paraffin embedding and subsequently tissues were sectioned at 5 mm. Slides were stained with hematoxylin and eosin (H&E). All the staining techniques will be performed as described by Bancroft and floyd 2013 (Bancroft and Floyd 2013).
2.5 | Blood sampling
Five mL of blood was collected by jugular vein puncture from the experimental sheep. The first. Meanwhile, the second blood sample was collected into a vacutainer tube containing ethylenediaminetetraacetic acid (EDTA) as an anti-coagulant, which was kept frozen at −80 °C until molecular analysis.
2.6 | Quantitative real-time PCR
Gene expression was quantified using the quantitative real-time PCR technique (qRT-PCR) for both more and less reactive sheep. All primers for genes encoding angiogenic proteins (NOS3, VEGF and HIF 1a, Table 1) were designed using Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Trizol reagent was used to extract RNA from peripheral blood cells (Puregene, Genetix brands). The RNA pellet was dissolved fully after being eluted with 50 L of RNase-free water and incubated for 10 minutes at 55°C. The extracted RNA was reverse transcribed to cDNA in a 20-liter reaction using the SensiFASTcDNA synthesis method (Bioline, London, U.K.), in which 5 μl of RNA sample were mixed with 4 liters of 5x TransAmp Buffer, 1 μl of reverse transcriptase enzyme, and 10 μl of UltraPureDNase/RNase-free water. Thermal cycling was used to incubate the reaction mixture at 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min. The cDNA samples were then diluted 1:10 in sterile DNase-free water and kept at -20°C for storage. 2 μl of cDNA template, 10 l SYBR Green PCR Master mix (SensiFAST SYBR NO- Imt kit, Bioline, London, UK), 0.8 ul of 10 μl forward and reverse primers (Vivantis Technologies Sdn Bhd., Malaysia), and 6.4 μl of sterilized Ultra-Pure DNase-free water were used to make a total volume of 20 ul. The reaction mixtures were heated to 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, followed by 72°C for 15 seconds. Gel electrophoresis and melting curve analyses were used to determine the specificity of each primer. Furthermore, the effectiveness of each primer was estimated using the formula 'Efficiency = 1+10(-1/slope)'. The 2Ct approach published by Livak and Schmittgen (Livak and Schmittgen 2001) was utilized to assess relative quantification of mRNA transcripts, with the -actin gene serving as the housekeeping gene.
2.7 | Statistical analysis
Appropriateness of fit for the normal distribution of model-residuals of measured parameters (TAMV, PI, RI, NOS3, VEGF, and HIF-1a) was visually assessed using normal probability plots (Q-Q-plots) and the Kolmogorov-Smirnov test. To determine the effect of time (wk) on TAMV, PI, RI on the expression of angiogenic proteins, a mixed model one-way analysis of variance (ANOVA), with time points as repeated measurements, was used. Post-hoc multiple pairwise comparisons were done according to a Tukey adjustment of error rate. Statistical analyses were conducted with commercial statistical software (SAS®, version 9.2, SAS Institute, Cary, NC). For all analyses, P ≤ 0.05 was defined as significant.