In Silico Analysis of miRNA-Mediated Genes in the Regulation of Dog Testes Development from Immature to Adult Form.

High-throughput in-silico techniques help us understand the role of individual proteins, protein-protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein-protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA's associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes' specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.

Impact of heat stress on embryonic development during first 16 days of gestation in dairy cows.

Objective was to elucidate the effects of heat stress (HS) on embryo development during first 16 gestational days (GD) and circulating hormone concentrations on GD-16 in lactating Holstein cows. Cows in HS and control (CON) groups were exposed to temperature humidity index (THI) of ≥ 73 and < 73, respectively, for 3 weeks before the experiment. GD-7 (67 vs 49%) and GD-16 (52 vs. 31%) conception rates following single insemination were greater (P < 0.01) for CON compared with HS cows. Control cows produced more GD-7 transferrable embryos following superovulation compared with HS cows (84.8 vs 53.1%; P < 0.001). Mean (± SEM) length (45.2 ± 10.6 vs. 59.2 ± 9.1 mm) and weight (31.4 ± 4.3 vs. 42.4 ± 6.2 mg) of GD-16 conceptus were greater for CON compared with HS cows (P < 0.05). Control cows yielded more filamentous conceptus (≥ 25 mm) compared with HS cows (71 vs 45%; P < 0.05). Progesterone (2.09-fold) was higher, and cortisol (1.86-fold), prolactin (1.60-fold), substance-P (1.55-fold), Isoprostane-8 (1.34-fold) and prostaglandin F metabolites (1.97-fold) were lower in CON compared with HS cows (P < 0.05). Progesterone positively, and substance-P, isoprostane-8 and the THI negatively were associated with GD-16 conceptus length (P < 0.05). In conclusion, altered hormones concentrations in heat-stressed cows plausibly resulted in lower GD-7 and GD-16 conception rates, fewer GD-7 transferable embryos, and stunted GD-16 conceptus elongation.

Association of gastrointestinal parasite burden, serum cytokines and hormones concentrations, and pregnancy in Angus-cross beef cows.

The objective was to elucidate the relationships among gastrointestinal (GI) parasite load, serum cytokines (Th 1 - Interleukin (IL) 2, Interferon (IFN) γ and Tumor necrosis factor (TNF) α; Th 2- IL4, IL6, and IL10) levels, hormones (progesterone, cortisol, 8-epi-prostaglandin F2 alpha (isoprostane), prolactin, substance-p, and prostaglandin F metabolites) concentrations, and pregnancy in beef cattle. Angus-cross beef cows (n = 700; age, 3-8 y) were blocked by age and body condition score (BCS, 1-9), and were randomly assigned to treatment (n = 350, TRT, 50 mg of eprinomectin/50 kg BW, im) or control (n = 350, CON, no treatment) on Day -30. Cows were synchronized using Controlled Internal Drug Release insert (CIDR) + CO-Synch protocol and artificially inseminated at a fixed time on Day 0 (66 h after CIDR removal). Fecal samples were collected to determine fecal egg count per gram (FEG, McMaster method) on Days -30, -23, -16, -7, 7, 0, 16 and 23, and blood samples were collected on Days -7, 0, 7, 16 and 23. Serum cytokines were determined on Days -7, 0, 7, 16 and 23, and circulating hormones were measured on Day 16. BCS were recorded on Day 16 following artificial insemination (AI), and pregnancy status was diagnosed on Day 30 and 60. Pregnancy/AI varied among treatment groups on Day 30 [TRT, 62.0% (217/350); CON, 54.9% (192/350) (P = 0.05)] and Day 60 [TRT, 60.9% (213/350); CON, 51.7% (181/350) (P < 0.05)]. Pregnancy loss between 30 and 60 days for TRT and CON groups were 1.8% (4/217) and 5.7% (11/192), respectively (P < 0.05). The BCS on Day 16 did not differ among treatment groups (P> 0.1). Four groups of 40 cows were selected based on their pregnancy status and treatment: pregnant, TRT; non-pregnant, TRT; pregnant, CON; and non-pregnant, CON to compare the mean FEG, cytokines, and hormones levels. The FEG and cytokine concentrations were significantly (P < 0.05) influenced by treatment, pregnancy status, day, treatment by pregnancy status, and treatment by day. Day 16 hormone concentrations were considerably influenced by treatment, pregnancy status, and treatment by pregnancy. Although FEG on Day -30 did not differ among the groups (P> 0.1), it was lower in treated, pregnant cows compared with cows in other three groups from Day -23 onwards (P < 0.05). Overall and pairwise comparisons showed that serum concentrations of Type 1 cytokines, IL2, IFNγ, and TNFα were lower (P < 0.05) from gestational Day 7 onwards in treated, pregnant cows compared with cows in other three groups. In contrast, serum concentrations of Type 2 cytokines, IL4, IL6 and IL10 were greater (P < 0.05) from gestational Day 7 onwards in treated, pregnant cows compared with cows in other groups. Serum concentrations of progesterone was greater and other hormones were lower for pregnant cows in TRT group compared to cows in other groups on gestational Day 16. In conclusion, GI parasite load was reduced; Th 1 cytokines levels were decreased; Th 2 cytokines concentrations were increased; progesterone level was increased; and cortisol, substance-P, prolactin, isoprostane, and PGFM were decreased in pregnant, TRT cows. These changes also resulted in an increase in P/AI. It is plausible that direct and bidirectional host-parasite interactions mediated by cytokines and hormones may have promoted maternal tolerance of an immunologically diverse conceptus and the establishment of pregnancy.

Day 7 embryo quality and suboptimal uterine environment influence morphometry of Day 16 conceptus in dairy cows.

The objectives of this study were to investigate the effect of Day 7 embryo quality and subclinical endometritis (SCE) in repeat breeder recipient cows on morphometry of Day 16 embryo and to determine the association of %PMN, serum progesterone and Day 16 conceptus length. Holstein dairy cows that failed to conceive at least 3 times, (parity, 3 and 4; body condition score, 3 to 3.5 out of 5) with subclinical endometritis (n = 180; SCE, >6% PMN on endometrial cytology) or without subclinical endometritis (n = 180; No-SCE, ≤ 6% PMN) were selected. Cows in each group received single, frozen-thawed, quality 1 (n = 60), 2 (n = 60) and 3 (n = 60) embryos (compact morula or early blastocyst) on Day 7 post estrus in the uterine horn ipsilateral to the ovary containing a corpus luteum, using standard nonsurgical techniques. Only cows that expressed estrus (Select-Synch protocol) and with acceptable corpus luteum (≥1.5 cm in size) were included. Conceptuses were collected on Day 16 from all recipient cows by standard non-surgical uterine flushing technique, using an 18-g embryo collection catheter with Phosphate Buffered Saline (pH 7.4). Blood samples were collected on Day 16 to determine serum progesterone concentrations. After collection, conceptuses were weighed and measured, and were categorized as tubular (underdeveloped, 10-20 mm) or filamentous (normal, >25 mm). Between cows with SCE and No-SCE, mean (±SEM) width (1.68 ± 0.13 mm vs. 1.84 ± 0.16 mm), length (34.4 ± 9.6 mm vs. 55.8 ± 13.4 mm) and weight (22.3 ± 3.7 vs. 40.6 ± 6.4 mg) of Day 16 conceptuses differed (P < 0.05). The mean width (1.75 ± 0.19 mm vs. 1.81 ± 0.22 mm), length (57.7 ± 11.2 vs. 51.1 ± 13.6 mm) and weight (34.3 ± 6.4 vs. 38.5 ± 8.2 mg) of Day 16 embryo following transfer of Day 7 embryo quality grade 1 and grade 2 embryos were not different (P > 0.1), but both differed from the mean width (1.59 ± 0.11 mm), length (28.9 ± 9.7 mm) and weight (25.3 ± 4.6 mg) of Day 16 embryo from Day 7 embryo quality grade 3 (P < 0.05). Total percentage of embryos recovered differed between SCE and No-SCE groups (P < 0.05; 36.1 vs 48.9%). Total percentage of embryos recovered on Day 16 following transfer of grade 1 (53.3%) and 2 (44.2%) Day 7 embryos were greater (P < 0.05) compared with transfer of grade 3 embryos (29.2%) (P < 0.001). Total percentage of filamentous embryos recovered was lower for SCE cows compared with No-SCE cows (P < 0.01; 15.0 vs. 25.6%). Total percentage of tubular embryos recovered did not differ between SCE and No-SCE cows (P > 0.1; 21.1% vs. 22.8%). Filamentous embryo recovered for grade 3 was lower (P < 0.05) compared with grade 1 in both SCE (8.3 vs. 21.7%) and No-SCE groups (15.0 vs. 33.3%). The mean (±SEM) CL volume (cm3; 11.8 ± 0.29 vs. 15.9 ± 0.31) and progesterone concentrations (ng/mL; 5.17 ± 1.8 vs. 8.2 ± 1.2) on Day 16 differed between SCE and No-SCE groups (P < 0.05) but not among Day 7 embryo grade groups (P > 0.1). The mean (±SEM) CL volume (cm3; 15.6 ± 0.28 vs 12.1 ± 3.9) and serum progesterone concentrations (ng/mL; 8.6 ± 1.4 vs. 4.9 ± 1.9) on Day 16 differed (P < 0.05) between cows yielded filamentous and tubular embryos. When all cows were considered, multiple regression analysis showed that the %PMN (P < 0.0001), progesterone concentrations (P < 0.0001), embryo qulaity (P < 0.05) and %PMN by progesterone interactions (P < 0.0001) influenced the length of Day 16 conceptus. Among cows without subclinical endometritis, only progesterone concentrations (P < 0.0001) and among cows with subclinical endometritis, only %PMN (P < 0.04) influenced the length of Day 16 conceptus. Progesterone concentrations (P < 0.0001) influenced the length of Day 16 conceptus in cows that received embryo quality 1 and 2. Progesterone concentration by %PMN interaction (P < 0.05) also influenced the length of Day 16 conceptus in cows that received embryo quality 2. The %PMN (P = 0.05) influenced the length of Day 16 conceptus in cows that received embryo quality 3. In conclusion, poor quality Day 7 embryo and presence of SCE negatively influenced early embryo development between Days 7 and 16 of gestation probably by dysregulated embryo-maternal interactions due to lower progesterone, prompting loss of the conceptus in sub-optimal uterine environment.

Estrous response and pregnancy percentages following use of a progesterone-based, split-time estrous synchronization treatment regimens in beef heifers.

Mean estrous response rate (%ERR) and pregnancy/AI percentages (%P/AI) were determined after imposing split-time AI (STAI) and fixed time AI (FTAI) following 14-d controlled internal drug release (CIDR)+PGF2α or 5-d Select Synch + CIDR regimens. In Experiment 1, 1152 heifers (five locations) were randomly assigned to 14- or 5-d and to 54 + 74- or 64 + 84-h STAI treatment combinations. Estrous detection patches were affixed at PGF2α administration (19 day after- and on day 5 at- CIDR removal for 14- and 5-d regimens, respectively), assessed at 54- or 64-h and again at 74- or 84-h after PGF2α. Heifers determined to be in estrus at respective times were inseminated and non-estrous heifers at 74- or 84-h were given GnRH and inseminated concomitantly. The %ERR between 54 + 74- and 64 + 84-h STAI combinations differed (73.2 % and 78.8 %, respectively; P < 0.05), but %P/AI did not. In Experiment 2, 2014 heifers (eight locations) were randomly assigned to 14- or 5-d regimens and were inseminated split-time (64+84-h combination, similar to Experiment 1) or at fixed time (72- or 56-h after PGF2α for 14- or 5-d regimens, respectively). There were differences (P < 0.01) between STAI and FTAI treatments for %ERR (81.3 % and 64.4 %) and %P/AI (61.2 % and 55.4 %). Estrous synchronization regimen by AI treatment interaction (P < 0.05) showed that the %ERR were 79.8 %, 82.6 %, 66.2 % and 62.8 % and the %P/AI were 58.9 %, 63.4 %, 56.5 % and 56.5 % (for 14-d/STAI, 5-d/STAI, 14-d/FTAI and 5-d/FTAI, respectively). In conclusion, the 5-d CIDR with 64+84-h STAI combination was the most effective because of the greater %P/AI when this regimen was imposed.

Presynchronization with CIDR, with or without GnRH, prior to CO-Synch in beef heifers.

Objectives were to compare ovarian responses and pregnancy per AI (P/AI) in Angus-cross beef heifers (n = 521; 4 locations) synchronized with CIDR-CO-Synch (CCOS) versus CIDR-GnRH-CO-Synch (CGCOS) protocols. Heifers were assigned a reproductive tract score (RTS: 1, immature, acyclic; 5, mature, cyclic), body condition score (BCS: 1, emaciated; 9, obese) and temperament score (0, calm, 1, excitable). Heifers in the CCOS (n = 261) group received a CIDR on Day -20 (removed on Day -13), 100 µg GnRH on Day -10, 25 mg PGF2α on Day -3 and were timed inseminated 60 h later, with concomitant GnRH (Day 0). Heifers in the CGCOS (n = 260) group received a CIDR on Day -26 (removed on Day -19), 100 µg of GnRH on days -16 and -10, 25 mg of PGF2α on Day -3 and were timed inseminated 60 h later, with concomitant GnRH (Day 0). Ovarian ultrasonography was done in a subset of heifers (n = 60; 30 in each group) to determine number and size of ovarian follicles and presence of corpus luteum (CL). There was increased (P < 0.05) percentage of heifers with CL in CGCOS group compared to heifers in CCOS group on Day -10 (82.3 vs 68.2%) and on Day -3 (88.3 vs 75.1%). Average size of the largest ovarian follicle on Day 0 was greater for heifers in CGCOS group compared to CCOS group (P < 0.05). However, P/AI did not differ between CCOS and CGCOS groups, 55.0% (143/260) and 59.8% (156/261), respectively (P > 0.1). In conclusion, CIDR presynchronization with or without GnRH (CCOS and CGCOS protocols) in beef heifers resulted in similar P/AI. Adding GnRH to presynchronization with CIDR resulted in more heifers with a CL at PGF2α and increased preovulatory follicle diameter at AI. Future studies are needed with bigger sample size and CIDR + CO-Synch treatment as control to determine economic benefit.

A Method to Isolate CD34+ Mononuclear Cells from Canine Peripheral Blood.

Embryonic stem cells are pluripotent whereas adult stem cells are multipotent in nature. In recent years, evidence suggests that adult stem cells not only differentiate into specific cell lineages but also transdifferentiate into multiple cell lineages. Progenitor cells are found in adult bone marrow, blood, and other organs and differentiate into numerous cell lineages regardless of origin. Identifying a subset that can differentiate into mature endothelial cells is essential. This article describes peripheral blood collection in adult beagle dogs, isolation of peripheral blood mononuclear cells (PBMNCs) from the cell fraction, separation of a subset of CD34+ cells using immunomagnetic principles, characterization of PBMNCs and CD34+ cells using flow cytometry, and evaluation of gene expression of CD34, KDR, and CD133 in CD34+ fractions. Efficient methods of isolation of endothelial progenitor cells (EPCs) will promote the ex vivo expansion and transplantation of EPCs in ischemic injury to enable neovascularization. © 2019 by John Wiley & Sons, Inc.

Fertility of Holstein heifers after two doses of PGF2α in 5-day CO-Synch progesterone-based synchronization protocol.

The objective of the study was to determine the effect of three different PGF2α (PGF) treatments in the 5-day CO-Synch progesterone-based synchronization protocol on artificial insemination (AI) pregnancy rate (PR) in Holstein heifers in Turkey and the United States. We hypothesized that two doses of PGF administered concurrently or 6 hours apart would result in greater AI pregnancy compared with a single dose of PGF on Day 5 at controlled internal drug release (CIDR) removal. In Turkey, Holstein heifers (n = 450) from one farm in the province of Adana and another farm in the province of Bursa were included. In the US, Holstein heifers (n = 483) from two locations in the state of Idaho were included. Heifers within locations were randomly allocated to one of three protocol groups: 1PGF-received 25 mg IM of dinoprost at CIDR removal; 2Co-PGF-received 50 mg IM of dinoprost at CIDR removal, and 2PGF-received 25 mg IM of dinoprost at CIDR removal and an additional 25 mg IM of dinoprost 6 hours later. All heifers received a CIDR (1.38 g of progesterone) and GnRH (10 µg IM of Buserelin [Turkey] or gonadorelin hydrochloride [US]) on Day 0. The CIDRs were removed on Day 5, and each heifer was given PGF according to the assigned treatments. On Day 7, each heifer was given another dose of GnRH and concurrently inseminated at 56 hours after CIDR removal. Heifers in both experiments were examined for pregnancy status between 35 and 45 days after AI. Overall, controlling for age, the heifers in the 2PGF group had greater AI-PR (61.7% [192/311]) than heifers in 2Co-PGF (48.2% [149/309]; P < 0.001) or 1PGF (53.7% [168/313]; P < 0.05) groups. No difference was observed between 2Co-PGF and 1PGF groups (P > 0.1). In Turkey, the heifers in the 2PGF group had a greater AI-PR (60% [90/150]) than 2Co-PGF (45.3% (68/150); P < 0.01] group. No difference was observed between 2PGF and 1PGF (55.3% [83/150]) groups (P > 0.1). There was a trend for AI pregnancy between 1PGF and 2Co-PGF groups (P = 0.08). In the United States, the heifers in the 2PGF group had a greater AI-PR (63.4% [102/161]) than the heifers in 2PGF (50.9 [81/159]; P < 0.05) or 1PGF (52.1% [85/163]; P < 0.05) groups. Heifers that were 15- and 16-month old achieved greater AI-PR than 17- and 18-month-old heifers (59.2 [342/578] vs. 47.0% [168/355]; P < 0.01). In conclusion, administration of 2PGF at 6 hours apart on Day 5 at CIDR removal in a 5-day CO-Synch + CIDR protocol resulted in greater AI pregnancy. A greater number of 15- and 16-month-old heifers became pregnant compared with 17- and 18-month-old heifers.

Fertility after two doses of PGF2α concurrently or at 6-hour interval on the day of CIDR removal in 5-day CO-Synch progesterone-based synchronization protocols in beef heifers.

Timed artificial insemination protocols in beef cattle are designed to synchronize ovulation in a greater proportion of females while simultaneously achieving acceptable pregnancy rates and a concise calving season. Protocols achieving such goals reduce time and labor associated with estrus detection and make advanced reproductive technologies implementable for beef producers. The objective of the study was to determine the effect of three different PGF2α (PGF) dosage schemes on artificial insemination (AI) pregnancy rates in beef heifers. We hypothesized that two doses of PGF administered concurrently at the time of controlled internal drug release (CIDR) removal would attain similar pregnancy rates compared with two doses given 6-hours apart-one at CIDR removal and the next 6 hours later in the 5-day CO-Synch progesterone-based synchronization protocol. Angus heifers (n = 875) at six locations in Washington, Idaho, and Oregon states were included in this study. Heifers within locations were assigned a body condition score (BCS). All heifers received a CIDR (1.38 g of progesterone) and 100 µg IM of GnRH on Day 0. The CIDRs were removed on Day 5, heifers were randomly allocated to one of three protocol groups: 1PGF (n = 291), received 25 mg IM of dinoprost (PGF); 2CO-PGF (n = 291), received 50 mg IM of dinoprost at CIDR removal, 2PGF (n = 293), received 25 mg IM of dinoprost at CIDR removal, and an additional 25 mg IM of dinoprost 6 hours later. Each heifer was given GnRH (100 µg, IM) and artificially inseminated at 56 hours after CIDR removal. Heifers were examined for pregnancy status between 50 and 70 days after AI to determine time of conception. A mixed-model procedure (PROC GLIMMIX of SAS) was used to evaluate the effect of treatments (1PGF, 2CO-PGF, and 2PGF) on AI pregnancy rates. Models included were treatments, BCS categories (≤5 and >5), and treatment by BCS category interaction. Location (state), handling facilities, handlers, inseminators, and AI sires were included as a random effect in the model. The 2PGF group had greater AI pregnancy rate of 63.6% (185/291), compared with the 2CO-PGF group at 51.9% (151/291) and 1PGF group at 54.9% (161/293; P < 0.001). An AI pregnancy rate of 50% (104/208) was observed for heifers with BCS less than or equal to 5 versus 58.9% (393/667) for heifers with BCS greater than 5 (P < 0.05). Location did not influence the AI pregnancy rate (P > 0.1). In conclusion, beef heifers received two 25-mg doses of PGF at 6-hour interval on Day 5 at CIDR insert removal in a 5-day CO-Synch + CIDR synchronization protocols achieved greater pregnancy compared with heifers received 50 mg of PGF concurrently at CIDR removal.

Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows.

BACKGROUND: Adipose tissue is an active endocrine organ which secretes a wide range of hormones and protein factors, collectively termed adipokines. Adipokines affect appetite and satiety, glucose and lipid metabolism, inflammation and immune functions. The objectives were to evaluate serum concentrations of adipokines (adiponectin, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6) in lactating dairy cows with postpartum uterine inflammatory conditions (metritis, clinical endometritis or subclinical endometritis) and in cows experiencing loss of body condition, and to assess the relationship of adipokines and body condition loss in the establishment of persistent uterine inflammatory conditions. METHODS: Lactating multiparous Holstein cows (N = 40), with body condition scores (BCS) from 2 to 4 (eight cows for each 0.5 score increment) were enrolled. Body condition was monitored for all cows weekly for 7 weeks post calving; cows with uterine inflammatory conditions were also re-evaluated 2 weeks later. Blood samples were collected from 1 week prior to calving to 7 weeks after calving for determination of serum concentrations of adipokines, insulin and insulin like growth factor (IGF)-1. RESULTS: Cows with metritis or clinical endometritis had higher serum concentrations of adiponectin, leptin, TNF-alpha, IL-1beta and IL-6 compared to normal cows (P < 0.05). Furthermore, serum leptin, TNF-alpha, IL-1beta and IL-6 were higher in cows with subclinical endometritis compared to normal cows (P < 0.05), and insulin and IGF-1 concentrations were lower in cows with metritis or clinical endometritis. Cows with low BCS (2 and 2.5) had significantly higher adiponectin, TNF-alpha, IL-1beta and IL-6 than those with high BCS (3 to 4). Cows with persistent uterine inflammatory conditions had higher adiponectin, leptin TNF-alpha, IL-1beta and IL-6 and insulin compared to normal and spontaneously recovered cows, except for IGF-1 (P < 0.05). CONCLUSIONS: Serum concentrations of adipokines, insulin, and IGF-1 had significant associations with BCS categories (low vs. high) and postpartum uterine inflammatory conditions. Perhaps loss of body condition mediated increases in anti- and pro-inflammatory cytokines, whereas increased pro- and anti-inflammatory cytokines concentrations mediated body condition loss and thereby prolonged persistence of uterine inflammation in dairy cows.

Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus.

BACKGROUND: Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. METHODS: Pregnant ewes (N=18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N=6) or 1,000 mg of gT (N=7) or placebo (CON; N=5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. RESULTS: Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P<0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P<0.05), whereas IL-1b was similar between treatment groups (P>0.1). CONCLUSIONS: Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8.

Tocopherol induced angiogenesis in placental vascular network in late pregnant ewes.

BACKGROUND: Tocopherols have biphasic, proangiogenic and antiangiogenic therapeutic effects. The objective of this clinical trial was to clarify tocopherol's placental angiogenic potential in late pregnant ewes following oral supplementation. METHODS: Eighteen pregnant ewes during late gestation were selected for this study. Ewes were given oral supplementation of 500 mg of alpha-tocopherol (aT; N=6) or 1000 mg of gamma-tocopherol (gT; N=7) or placebo (CON; N=5) once daily from 107 to 137 days post breeding. Serum was obtained at weekly intervals and tissue samples were obtained at the end of supplementation to: 1) evaluate tocopherol concentrations in serum, uterus and placentome; 2) evaluate relative mRNA expressions of Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PlGF), endothelial Nitric Oxide Synthase (eNOS) and Hypoxia Inducible Factors (HIF) in uterus, caruncle and cotyledon; 3) analyze the morphometry of the placental vascular network. RESULTS: Supplementation of aT or gT resulted in increased concentrations in serum, placentome and uterus compared to control (P<0.05). In aT group, mRNA expressions of PlGF, eNOS and HIF-1alpha in cotyledon were greater than the CON group. In gT group, mRNA expressions of VEGF, eNOS, HIF-1 alpha and HIF-2 alpha in caruncle and uterus, and HIF-1alpha in cotyledon, were greater than the CON group. Morphometry analysis revealed increased angiogenesis in the supplemented groups. CONCLUSION: Daily oral supplementation of aT or gT increased angiogenesis in the placental vascular network in pregnant ewes during late gestation. Increase in placental angiogenesis may provide nutrients required for the development and growth of fetus during late pregnancy.