29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
L. B. Ferré ◽  
C. Fresno ◽  
M. E. Kjelland ◽  
P. J. Ross

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although >80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

2015 ◽  
Vol 27 (1) ◽  
pp. 194
Author(s):  
S. Saugandhika ◽  
H. N. Malik ◽  
S. Saini ◽  
V. Sharma ◽  
S. Bag ◽  
...  

Interferon tau (IFN-tau) is known as maternal pregnancy recognition factor in ruminants. IFN-tau not only acts as a signalling molecule of pregnancy recognition but also performs various functions for successful implantation and pregnancy establishment. The aim of the present study was to produce recombinant buffalo interferon-tau (BuIFN-Tau) and observe if it has any effect on in vitro embryo development. The BuIFN-Tau gene was obtained through polymerase chain reaction (PCR) from hatched buffalo blastocysts and was cloned into pJET cloning vector. Screening of the recombinant colonies gave 8 distinct buffalo IFN-tau isoforms, out of which the predominant buffalo IFN-t tau1 isoform (gene bank accession number JX481984), was subcloned into expression vector pET22b without signal sequence. The recombinant plasmid was induced to express the recombinant protein by isopropyl b-D-1-thiogalactopyranoside. Analysis of the products of recombinant BuIFN-tau without signal sequence by SDS–PAGE revealed a new 20-kDa protein coinciding with the molecular weight of IFN-tau as reported earlier in literature. The purified recombinant BuIFN-tau was confirmed by Western blot using anti-HIS antibody and was subjected to three steps of large-scale purification using HIS affinity chromatography, anion exchange chromatography, and gel filtration chromatography. Finally, a relatively pure histidine-tagged recombinant protein, which had a purity of at least 90%, was generated as confirmed through SDS. The concentration of recombinant BuIFN-tau was 1 mg mL–1 by Bradford assay. The purified recombinant BuIFN-tau was used as supplement of the culture medium for IVF early buffalo embryos at the following concentrations: control, 1, 2, and 4 µg mL–1. Sixty oocytes each in 4 groups (with 20 oocytes/drop in three replicates for each group) were used for in vitro maturation. After 24 h, the matured oocytes were incubated with in vitro capacitated sperm cells for 18 h; thereafter, the presumptive zygotes were cultured in IVC medium supplemented with 0, 1, 2, or 4 µg mL–1 of the purified recombinant BuIFN-tau. The experiment was repeated 3 times. The data were analysed using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values. The differences were analysed by one-way ANOVA followed by Fisher's least significant difference test. Out of 3 concentrations of recombinant BuIFN-tau, the 2 µg mL–1 concentration significantly promoted the rate of blastocyst development, 45.55% against 31.1% (control; P < 0.01). Blastocyst development rate for low and high concentrations was 29.97% and 10.18% respectively. It is concluded that the addition of 2 µg mL–1 of recombinant BuIFN-tau enhances the blastocyst development rate in buffalo, and hence there is some evidence that BuIFN-tau has not only a role in maternal recognition of pregnancy but also in embryonic development.


Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 173-180 ◽  
Author(s):  
M. Benkhalifa ◽  
A. Demirol ◽  
T. Sari ◽  
E. Balashova ◽  
M. Tsouroupaki ◽  
...  

SummaryIn repeated implantation failure, the co-culture of human embryos with somatic cells has been reported to promote the improvement of embryos quality, implantation and pregnancy rate. It was reported that feeder cells can be more beneficial to the oocyte and embryo by detoxifying the culture medium and supporting embryo development via different pathways. In this study, 432 patients, each with a minimum of three repeated implantation failures, were accepted for a prospective randomized study with or without autologous cumulus cell embryo co-culture and transfer at day 3 or day 5–6. We also investigated the expression of leukaemia inhibitor factor (LIF) and platelet activating factor receptor (PAF-R) on day 3 confluent cumulus cells. The statistic analysis of the data showed significant difference of implantation and clinical pregnancy rates between classical culture and day 3 compared with co-culture and day 5–6 transfer. The molecular analysis showed that cumulus cells express the LIF and the PAF-R genes and confirmed the possible positive role of growth factors and cytokines in early embryo development. Embryo co-culture systems with autologous cells can be beneficial in routine in vitro fertilization for embryo selection and implantation improvement. More molecular investigations need to be done to improve elucidation of the complex dialogue between the embryo and feeder cells prior to implantation and to understand the involved biological function and molecular process during embryo development.


2012 ◽  
Vol 24 (8) ◽  
pp. 1084 ◽  
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
A. Langbeen ◽  
E. P. A. Jorssen ◽  
E. Bosmans ◽  
...  

Producing bovine in vitro embryos individually is a challenge as it generally leads to impaired embryo development. Earlier research optimised a single embryo in vitro production (IVP) protocol using serum, cumulus cells and oil during culture. As some of these factors are undesirable in certain circumstances, the present study investigated their necessity and possible interactions, and defined their role during single-embryo culture. Although the cumulus cell monolayer produced progesterone, it appeared not to be a key factor in supporting single-embryo development. Because in vitro culture in large medium volumes was shown to impair single-embryo development, two new oil-free culture protocols were tested. Using a 30-µL droplet of medium in 96-well plates with a small surface area resulted in comparable blastocyst rates to those obtained under oil. When serum was used, co-culture with cumulus cells seems necessary, leading to consistently high blastocyst rates. Finally, a serum-free, oil-free culture system using insulin, transferrin, selenium and BSA resulted in embryos with similar total cell numbers and apoptotic cell ratios, but blastocyst rates did not equal those obtained with serum and co-culture. This research additionally stresses the fact that specific interaction mechanisms between somatic cells and a developing in vitro embryo are far from unravelled.


2015 ◽  
Vol 63 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Fotini Krania ◽  
Eleni Dovolou ◽  
Constantinos A. Rekkas ◽  
Sonia Heras ◽  
Ioannis Pappas ◽  
...  

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.


2016 ◽  
Vol 65 (1) ◽  
Author(s):  
F. Frati ◽  
C. Incorvaia ◽  
F. Marcucci ◽  
L. Sensi ◽  
G. Di Cara ◽  
...  

Sublingual immunotherapy (SLIT) currently represents, as indicated by meta-analysis of its efficacy and safety, a valid option to the generally used traditional subcutaneous immunotherapy (SCIT) for treating respiratory allergy. Regarding efficacy, recent studies demonstrated that, similar to what has already been observed in SCIT as well as in experimental and clinical studies about the magnitudo of allergen exposure, the effectiveness on both clinical symptoms and immunologic changes depends on the amount of allergen administered during treatment. In addition, in vitro studies addressed with the role of dendritic cells, currently considered to be of pivotal importance in orienting toward tolerance the immune response to allergens, showed that the internalisation of allergen molecules, which is followed by tolerogenic presentation to T cells, depends on the amount of allergen. However, such dose dependence is not apparent concerning the safety. In fact, the comparison of studies respectively conducted with high and low allergen doses did not show differences in the rate of systemic reactions, which in any case never had the presentation of anaphylaxis, and instead a significant difference in the rate of local reactions, following the oral and gastrointestinal contact with the allergen extract, in favour of high dose studies.


2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 93-93
Author(s):  
Sara Ebarb ◽  
Sabrina May ◽  
Mark D Newcomb

Abstract Fiber ingredients in swine diets have various components that affect the intestinal tract distinctively. The objective of this study was to determine the effect of structural fiber sources on growth performance of nursery pigs. A total of 1,760 weanling pigs (initial BW = 6.12 ± 0.3 kg, 16 reps/trt, 22 pigs/pen) were used in a study with 5 dietary treatments: 1) Complex nursery diet with no additional fiber (CON); 2) CON + 2% rice hulls; 3) CON + 4% rice hulls; 4) CON + 6% wheat middlings; 5) CON + 12% wheat middlings. Fiber ingredients were added at the expense of corn and some processed soybean meal to maintain similar dietary protein levels. Using an in vitro fermentation estimation of fermentation for rice hulls and wheat middlings, diets were constructed to add similar levels of structural (non-fermentable NDF) fiber between low and high additions of rice hulls and wheat middlings. Pens across two barns were randomly allotted independent of one another and within a location block to one of the five treatments on d 0 of a two-phase study (d 0–11 and d 11–20.5 post-weaning). Data were analyzed by general linear model in R. Mortality and removal data were analyzed as a generalized linear mixed model with a binomial distribution. Contrasts tested the effect of additional fiber (CON vs treatments 2–5), effect of medium vs high fiber (treatment 2/4 vs 3/5), effect of source of fiber (treatment 2/3 vs 4/5), and the interaction of level and source of fiber. Overall (Table 1), additional fiber resulted in increased ADFI (P &lt; 0.05) and tended to increase ADG (P &lt; 0.10). Probability of mortality and removal was reduced (P &lt; 0.05) when additional fiber was included. In summary, increasing the level of structural fiber improved performance and livability of nursery pigs.


Author(s):  
Congying An ◽  
Jinglan Liu ◽  
Qiaohui Liu ◽  
Yuqi Liu ◽  
Xiaoli Fan ◽  
...  

A growing number of studies suggest that the perceived sensory dimensions (PSDs) of green space are associated with stress restoration offered by restorative environment. However, there is little known about PSDs and stress restoration as well as their relationship to forest park. To fill this gap, an on-site questionnaire survey was conducted in three forest parks in Beijing, as a result of which a total number of 432 completed responses were collected and analyzed. The mean values of PSDs were used to represent PSDs of forest park. Using independent sample t-test and ANOVA, this study analyzed the individual characteristics that affected PSDs and stress restoration. Linear mixed model was used to identify the relationship between PSDs and stress restoration of forest park, which took into account the interactions of stress level and PSDs. The results showed that: (1) the perceived degree of PSDs in forest park from strong to weak was Serene, Space, Nature, Rich in species, Prospect, Refuge, Social and Culture, which varied with visitors’ gender, age, level of stress, visit frequency, activity intensity, visit duration and commuting time; (2) in PSDs, Refuge, Serene, Social and Prospect had significantly positive effects on the stress restoration of forest parks (3) there was no significant difference in the effect of the eight PSDs on the stress restoration between different stress groups; (4) stress restoration was influenced by visitors’ gender, age, visit frequency and visit duration. These findings can offer references for managers to improve the health benefits of forest park for visitors, and can enrich the knowledge about PSDs and stress restoration.


Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.


2021 ◽  
Author(s):  
Samat Amat ◽  
Devin B Holman ◽  
Kaycie Schmidt ◽  
Ana Clara B Menezes ◽  
Friederike Baumgaertner ◽  
...  

Emerging evidence has indicated that microbial transmission from the bovine dam to her fetus may take place before birth, and that the maternal microbiota during pregnancy modulates programming of fetal metabolic and nervous system development, highlighting the potential and extended role of the maternal microbiome in calf health and development. In the present study, we characterized the nasopharyngeal, ruminal and vaginal microbiota from two cohorts of beef heifers managed at the same location: 1) virgin yearling heifers (9 months old) born from dams received gestational diets which resulted in low (LG, n = 22) or medium (MG, n = 23) weight gain during the first 84 days of gestation; and 2) pregnant replacement heifers that received a vitamin and mineral supplement (VTM, n = 17) or not (Control, n = 15) during the first 6 months of gestation. Nasopharyngeal and vaginal swabs as well as ruminal fluid were collected from both cohorts and the microbiota of each sample was assessed using 16S rRNA gene sequencing. In addition to the comparison between treatment groups within each cohort, the similarity of the microbiota of the three sample types were evaluated, and shared taxa amongst these communities were identified. The bacterial genera present in the rumen and vagina that can influence methanogenic archaeal genera were predicted using a stepwise-selected generalized linear mixed model. No significant difference was observed in the alpha and beta diversity in any of the nasopharyngeal, ruminal and vaginal microbiota between LG and MG offspring virgin heifers, or between the control and VTM pregnant heifers (p > 0.05). Subtle compositional changes in the vaginal microbiota in yearling heifers, and in the nasopharyngeal and ruminal microbiota of pregnant heifers were detected in response to treatments. Forty-one archaeal and bacterial OTUs were shared by over 60% of all samples from both virgin and pregnant heifers. Two taxa within the Methanobrevibacter genus were identified as core taxa and this genus was more relatively abundant in pregnant heifers compared to virgin heifers. Among the 25 top genera, Prevotella and Prevotella UCG-003 (negative) and Christensenellaceae R-7 group (positive) were predicted to have a significant effect on ruminal Methanobrevibacter spp. The results of this study indicate that there is little impact of divergent gestational nutrition during the first trimester on the calf microbiome at 9 months postnatal, and that VTM supplementation during pregnancy may not alter the maternal microbiome. This study provides evidence that there are several microbial taxa, including methanogenic archaea, that are shared across the respiratory, gastrointestinal, and reproductive tracts, suggesting the need for a holistic evaluation of the bovine microbiota when considering potential maternal sources for seeding calves with pioneer microbiota.


Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Susan Alderman ◽  
Terri Armstrong ◽  
Geri Wood ◽  
Sean Savitz ◽  
Joshua Breier

Background: One year after stroke 50-70% of survivors have information processing speed (IPS) impairment. IPS is a complex, elemental cognitive function with close but separate relationships with memory, attention, executive function, reading, and writing. IPS impairment can be devastating, even without physical deficits. Hypothesis: The frequency and severity of IPS impairment in patients with acute stroke is unrelated to stroke severity. Methods: In a longitudinal study, 30 adults with acute mild (NIHSS <5) versus moderate stroke (NIHSS 5-12) were assessed for IPS and memory impairments at three time points (< 72 hours, Week 3, and Week 12). The Symbol Digit Modalities Test (SDMT) was the primary IPS instrument, scores below the norm (51-62 points) indicate impairment. Results: Using linear mixed model regression, no significant difference was noted between patients with mild and moderate stroke in frequency and severity of IPS impairment (p=0.2). All patients had baseline abnormal SDMT scores, improving 21% by Week 12 (p=.005) with one patient scoring as unimpaired (51 points). Patients with mild stroke (n=18) had baseline scores on average 53% lower than SDMT norm (mean score 24 (12), [95% CI] 6 [18, 30], Standard Error (SEM) 3) and 32% lower at Week 12 (mean 34 (11), [95% CI] 5 [29, 40], SEM 3). Patients with moderate stroke (n=12) had baseline SDMT scores 60% lower than norm (mean 21 (11), [95% CI] 6 [15, 27], SEM 3) and 39% lower by Week 12 (mean 31 (12), [95% CI] 7 [25, 38], SEM 3). Memory impairment at enrollment (<72 hours post stroke) was noted in 100% of patients with moderate stroke and 78% of patients with mild stroke; at Week 12 the frequency of memory impairment was equal in both groups (67%). Conclusion: IPS were critically impaired, in both mild and moderate stroke, with minimal spontaneous recovery at Week 12. IPS impairment is clinically significant during stroke recovery. IPS impairment should be considered in the planning of healthcare of stroke victims even with mild stroke.


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