Hormonal protocol used for cervical dilation in ewes does not affect morphological embryo quality but reduces recovery rate and temporarily alters gene expression

2021 ◽  
Author(s):  
Juliana Dantas Rodrigues Santos ◽  
Ribrio Ivan Tavares Pereira Batista ◽  
Rodolfo Ungerfeld ◽  
Augusto Ryonosuke Taira ◽  
Caroline Gomes do Espírito Santo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Recovery Rate ◽  
Embryo Quality ◽  
Cervical Dilation

scholarly journals Investigating the Intrafollicular Factors Affecting Oocyte Developmental Competency

2021 ◽  
Author(s):  
◽  
Zaramasina Clark
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Significant Finding ◽  
High Quality ◽  
Final Maturation

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos.  The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes.  The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation.  The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>

Proteinase-activated receptors in ovine cervical function

2005 ◽  
Vol 17 (7) ◽  
pp. 693 ◽  
Author(s):  
Sharon E. Mitchell ◽  
John J. Robinson ◽  
Margaret E. King ◽  
Lynda M. Williams
Keyword(s):  
Gene Expression ◽  
In Situ Hybridisation ◽  
Prostaglandin F2α ◽  
Cervical Dilation ◽  
Proteinase Activated Receptors ◽  
Pregnant Ewe ◽  
High Level ◽  
And Function

In sheep, inflammation not only functions in cervical dilation at parturition, but also plays an important part in the non-pregnant ewe cervix, as demonstrated by the high level of expression of interleukin (IL)-8 at oestrus. Ewes artificially induced to ovulate have significantly lower levels of IL-8 gene expression at oestrus compared with natural oestrus, indicating an inhibition of inflammation and function, offering an explanation for the low rates of conception in vaginally inseminated synchronised ewes. To identify potential pro-inflammatory agents to combat the anti-inflammatory effects of hormonal synchronisation of oestrus, we have investigated the role of proteinase-activated receptor (PAR)-1 and PAR-2. To localise and measure the level of expression of these receptors, ovine-specific probes were derived for PAR-1 and PAR-2 and used for quantitative in situ hybridisation in the ovine cervix. Both PAR-1 and PAR-2 were expressed in the luminal epithelium of the cervix throughout the oestrous cycle, with expression being highest at oestrus. The gene expression of PAR-2 at oestrus was approximately 30% higher than that of PAR-1. Artificial synchronisation of oestrus by either an intravaginal progesterone sponge or prostaglandin F2α injections did not inhibit PAR-1 or PAR-2 expression at oestrus; rather, in the case of PAR-2, progesterone synchronisation increased it. Both synchronising procedures increased the expression of PAR-1 and PAR-2 during the luteal phase of the cycle. Therefore, agonists of PAR-1 and PAR-2 may be potentially useful pro-inflammatory agents countering the inhibition of inflammation by hormonal synchronisation.

scholarly journals Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1967
Author(s):  
J. Dorado ◽  
M. Bottrel ◽  
I. Ortiz ◽  
M. Díaz-Jiménez ◽  
B. Pereira ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Embryo Transfer ◽  
Recovery Rate ◽  
Embryo Quality ◽  
Donor Age ◽  
Successive Cycle ◽  
Assisted Reproductive Technique ◽  
Factors Affecting ◽  
Embryo Recovery ◽  
Starting Point

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

Efficiency of Leukemic Stem Cell Separation From Patients with Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4997-4997
Author(s):  
Lu Zhang ◽  
Susanne Hofmann ◽  
Lars Bullinger ◽  
Marlies Goetz ◽  
Markus Wiesneth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Sorting ◽  
Peripheral Blood ◽  
Recovery Rate ◽  
Mononuclear Cells ◽  
Separation Efficiency ◽  
Conflicts Of Interest ◽  
Cell Number ◽  
Hematopoietic Stem

Abstract Abstract 4997 Leukemic stem cells (LSC) are the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSCs probably represent a critical target for therapeutic options. Xenograft models confirmed repeatedly that LSCs from AML patients reside mainly in CD34+CD38- compartment of leukemic blasts which makes the pure and efficient separation of this population mandatory to identify new therapeutic drugs to target LSC in different AML subtypes. We separated this subpopulation out of primary AML peripheral blood mononuclear cells (PBMC) samples with fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) and compared the efficiency of both methods. In order to profile gene expression of LSCs and hematopoietic stem cells (HSC) MicroArrays were performed using GeneChip Human Genome U133 Plus 2.0 from Affymetrix. The CD34+CD38- subpopulation was separated from PBMCs of 12 AML patients and 5 healthy volunteers using FACS. Concerning the 12 primary AML samples, the ratio of CD34+CD38- cells ranges between 0.79% and 86.2% using 1–5×107 PBMC for separation. After sorting, the purity of those AML samples increased to 88.4–98.4% while 2×104-3.6×106 cells were obtained. MACS was used to separate 2 representative samples, in which the CD34+CD38- subpopulation was rather small (sample1: 0.78%) or large (sample2: 86.1%). Those sorted subpopulations were compared to the samples sorted via FACS. In order to evaluate separation efficiency in a standardized manner, we defined the recovery rate: (CD34+CD38- cell number obtained /total CD34+CD38- cell number) × 100%. The total CD34+CD38- cell number was calculated through a pre-sorting FACS analysis. For sample 1, MACS resulted in a recovery rate of 4.2–6.4% with a purity of 86.6–90.3%, which is inferior to the recovery rate of 17% and the purity of 92.1% using FACS. For Sample 2, MACS resulted in a recovery rate of 0.4% with a purity of 98.8%, compared to the recovery rate of 11.6% with a purity of 98.1% by FACS. Comparing both methods it is obvious that the purity doesn't differ a lot, but the yield is much higher using FACS. This could represent a powerful tool, when managing rare samples. Finally, by comparing purity and yield, we showed that FACS is the adequate separation method. At the moment MicroArrays are being performed in order to investigate the gene expression profile for 12–15 AML patients and 5 HVs. Taken together, we showed a widely efficient method to routinely separate LSCs from patients with different subtypes of AML. Microarrays, that have been performed, represent a method that allows the comparison of the characteristics of LSCs in different AML subtypes and also of LSCs from bone-marrow with LSCs from peripheral blood and with HVs. These array data analyses are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

240 RELATIONSHIP BETWEEN PEAK NUMBER OF ANTRAL FOLLICLES AND FOLLICULAR WAVES, HORMONE CONCENTRATIONS, SUPEROVULATORY RESPONSE, AND EMBRYO QUALITY IN BEEF HEIFERS

2006 ◽  
Vol 18 (2) ◽  
pp. 228 ◽  
Author(s):  
F. Ward ◽  
P. Lonergan ◽  
F. Jimenez-Krassel ◽  
J. J. Ireland ◽  
A. C. O. Evans
Keyword(s):  
Recovery Rate ◽  
Embryo Quality ◽  
Corpora Lutea ◽  
Beef Heifers ◽  
High Group ◽  
Estrous Cycles ◽  
Antral Follicles ◽  
Peak Number ◽  
Follicular Waves ◽  
The Mean

Recent studies in dairy cattle demonstrate that the number of antral follicles during follicular waves is highly variable among animals, but highly repeatable within individuals, and the numbers of follicles during waves are inversely associated with serum FSH concentrations (Burns et al. 2005 Biol. Reprod. 73, 54-62). The aims of this study were to determine (1) serum FSH and estradiol concentrations during follicular waves in beef heifers categorized according to peak number of antral follicles per wave, and (2) the relationship between this categorization and superovulatory response. Estrous cycles of crossbred beef heifers (n = 90) were synchronized with two i.m. injections of PGF2� 11 days apart; animals were scanned daily for 5 days beginning 24 h after the second PG injection to determine the number of follicles during the first follicle wave in two or three consecutive estrous cycles. Heifers were grouped based on the peak number of follicles e 3 mm per wave in diameter (Low: d 15 follicles, n = 12; High: e 25 follicles, n = 11). Intermediate animals were excluded. Heifers were then re-synchronized and blood samples taken every 8 h up to Day 7 to characterize FSH and estradiol profiles. Subsequently, animals were superovulated (pFSH, Folltropin�; Bioniche Animal Health, Belleville, Ontario, Canada), inseminated, and flushed on Day 7; this procedure was repeated twice. All values for hormone concentrations were aligned relative to the peak FSH value. A mixed model, repeated-measures approach was used to determine if serum FSH and estradiol concentrations were different between groups. Differences in recovery rate (number of oocytes/embryos recovered compared to number of corpora lutea) and proportion of transferable embryos were analyzed using Chi-square analysis. The mean (�SE) peak postovulatory FSH concentration was lower (P d 0.03) for animals with high numbers of follicles. No differences in estradiol concentrations were observed. The mean superovulatory response (number of corpora lutea) was higher (P d 0.05) in the High group than in the low group (17.6 � 3.6 vs. 8.5 � 1.1). While there was no difference in the recovery rate of embryos (62 vs. 60%), significantly more (P d 0.05) oocytes/embryos (10.6 � 2.7 vs. 4.7 � 0.7) and transferable embryos (5.4 � 1.3 vs. 3.8 � 0.8) were recovered per animal in the High group. Of the embryos recovered, the proportion of transferable quality was higher for animals in the Low group (80 vs. 51%; P d 0.05). In conclusion, the number of antral follicles during a follicular wave in beef heifers is inversely related to peak postovulatory FSH concentration, but is unrelated to estradiol concentration. In addition, while the superovulatory response and number of transferable embryos were greater in animals with a high number of antral follicles, embryo quality, measured as the proportion of transferable embryos per donor, was reduced. We conclude that cattle with relatively high numbers of follicles per wave respond best to standard superovulation protocols. However, the reason the proportion of high quality embryos is reduced in cattle with high vs. low numbers of follicles per wave is unknown.

183 EXERCISE INFLUENCES EMBRYO QUALITY IN MARES

2010 ◽  
Vol 22 (1) ◽  
pp. 250 ◽  
Author(s):  
R. Smith ◽  
K. Vernon ◽  
J. Gibbons ◽  
D. Kelley ◽  
J. Anton ◽  
...  
Keyword(s):  
Recovery Rate ◽  
Embryo Quality ◽  
Exercise Group ◽  
Quality Score ◽  
Control Group ◽  
Recovery Rates ◽  
Embryo Recovery ◽  
Humid Environment ◽  
Negative Effect ◽  
The Mean

Athletic mares are commonly utilized in an embryo transfer regimen during the breeding season, which allows owners to continue to train and compete with these animals. Exercising mares in a hot and humid environment has proven detrimental to embryo recovery rates. However, the effects of exercise under milder temperatures have not been examined. Our goal was to compare embryo recovery rates and embryo quality scores of mares under mild environmental conditions of 2 differing exercise regimens v. a single control group (n = 5). One treatment group (full exercise) consisted of mares (n = 5) exercised 6 days a week throughout the duration of the project. The partial-exercise group (n = 5) was exercised throughout the duration of the project 7 days a week, with full rest from day of detected ovulation to the proceeding embryo collection attempt. This was to limit any deleterious effects exercise may potentially have on the mare and subsequent early developing equine embryo. Exercise resumed the day after an embryo collection attempt for the partial-exercise group. Exercised mares were given a 1-month training period prior to the beginning of this project and were moderately exercised for 30 min in a mechanical exercise pen (free exerciser). Control mares were managed similarly as the other groups, however were not exercised. Mares were bred via artificial insemination and embryos were nonsurgically collected 7 d post-ovulation. Mean environmental temperature during the duration of this study was 27.1°C with average 60% humidity. Immediately following completion of exercise, mares in the full exercise group had a mean temperature increase of 1.0°C and partial-exercised mares increased 0.9°C. Exercise had a significant effect on embryo quality (P < 0.05, Table 1) but did not influence embryo recovery rate. The mean embryo quality score for the control group was 1.1, whereas the full-exercised group was 1.85 and partial-exercised group was 2.5. It appears from these data that exercise has a negative effect on embryo quality, thus lowering the availability of transferable embryos. Embryos of lower quality (≥3) have been shown to result in poor pregnancy rates in horses. Additionally, rest from ovulation to an embryo collection attempt did not improve embryo quality. Table 1.Embryo recovery rate and quality score of embryos from control and exercised mares

Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos

2015 ◽  
Vol 27 (7) ◽  
pp. 1115 ◽  
Author(s):  
Miriam Castillo-Martín ◽  
Marc Yeste ◽  
Albert Soler ◽  
Roser Morató ◽  
Sergi Bonet
Keyword(s):  
Gene Expression ◽  
Ascorbic Acid ◽  
Dna Fragmentation ◽  
Embryo Quality ◽  
Survival Rates ◽  
Transcript Abundance ◽  
Cell Number ◽  
Ascorbic Acid Supplementation

The aim of the present study was to determine the effect of l-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification–warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without l-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without l-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with l-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with l-ascorbic acid. Thus, supplementing culture and/or vitrification media with l-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.

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