Improve Embryo Quality Research Articles

Comparison of Intracytoplasmic Sperm Injection Outcome with Sperm Selection Techniques in Oligoasthenozoospermic Males: A Randomized Controlled Trial

Background: Male infertility rate has increased over the recent years across the globe. Intracytoplasmic sperm injection (ICSI) is mainly used to treat male infertility; however, in this method, sperm selection plays a critical role in improving ICSI outcome. Objectives: The aim of this study was to evaluate the efficiency of three sperm selection techniques on ICSI outcomes in oligoasthenozoospermic men. Methods: This randomized clinical trial study was performed on 80 oligoasthenozoospermic (OAS) couples receiving ICSI in Milad Infertility Center, Mashhad, Iran from October 2016 to June 2017. The couples were randomly assigned to the three groups of control, hypo-osmotic swelling test (HOST) and hyaluronic acid (HA) according to the sperm selection technique for ICSI. Fertilization rate, embryo development (ED), embryo quality (EQ) and implantation rate (IR) were evaluated and compared among the groups. Results: Our results showed that there was no significant difference in fertilization rate between the studied groups. ED rate was increased in the HOST and HA groups compared to the control group, but this difference was not significant. EQ was increased in the HOST (P = 0.001) and HA groups (P = 0.001) compared to the control group. IR was enhanced in the HA group in comparison with the control group (P = 0.021), but there was no significant difference between the HOST and HA groups. Conclusions: The results of this study showed that for achieving successful clinical outcomes and improved embryo quality and implantation rate in oligoasthenozoospermic males undergoing Intracytoplasmic sperm injection, effective sperm selection through either the HOST or HA technique is necessary.

Role of melatonin on embryo viability in sheep.

Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day-night perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes (fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function, reduced antiluteolytic mechanisms, or improved embryo quality. This review focuses on the effects of melatonin on embryo viability and summarises the processes by which this hormone affects the ovary, follicle, oocyte, corpus luteum and embryo. Moreover, the effects of melatonin on the mechanisms of invivo maternal recognition of pregnancy in sheep and the protective action that it appears to have on the invitro procedures that are used to obtain healthy embryos are reviewed.

193 Melatonin implants in spring improve embryo production of aged ewes after superovulation regardless of endometrial progesterone receptor expression

Twenty-three Rasa Aragonesa aged ewes (average age: 10.3±0.3 years) were used to determine the effect of melatonin on ovulatory response, embryo production, and endometrial expression of progesterone receptors (PR) after superovulation. Ewes were treated (M, n=13) or not (control, C, n=10) with melatonin implants in March (Day 0, Northern Hemisphere autumn), and received intravaginal progestogen sponges for 14 days on Day 77. Superovulatory treatments consisted of 8 doses in decreasing concentrations (2 mL×2 and 1 mL×6) of 176 NIH-FSH-S1 units of NIADDK-oFSH-17 (Ovagen, ICPbio Reproduction, Auckland, New Zealand) administered twice daily starting 72h before sponge removal. Seven days after oestrus, embryos were recovered by laparotomy, ewes were killed, and uterine horns were processed to study PR expression by immunohistochemistry. The amount of PR was estimated subjectively by 2 independent observers in 5 endometrial compartments: luminal epithelium (LE), superficial (sGE) and deep (dGE) glandular epithelia, and superficial (sS) and deep (dS) stroma. The extent of staining was expressed on a scale from 0 to 100. Data were analysed with a 2×2 factorial ANOVA. Melatonin implants improved fertilization (92v. 57%, for M and C groups, respectively; P<0.01), blastocyst (47v. 9%; P<0.01), viability (88v. 31%; P<0.0001), and freezability (69v. 21%; P<0.001) rates. Specifically, melatonin induced a significant reduction of the number of non-viable (degenerate and retarded) embryos (0.3v. 1.5; P<0.05) and increased blastocysts (2.8v. 0.8; P<0.05) per ewe. Melatonin treatment decreased PR staining intensity (47v. 55%; P<0.05), but this effect was not observed when the individual cell types were compared (Table 1). Because the number of corpora lutea (CL) was responsible for different PR expression in both groups (P<0.0001), animals were divided into 2 ovulation rate categories: <10 CL and ≥10 CL, with lesser PR expression in the ≥10 CL group (P<0.0001); this lower PR immunostaining in ≥10 CL is consistent with progesterone down-regulation of its own receptor. An interaction among number of CL and treatment was found for embryo quality (P<0.05); thus, the positive effect of melatonin on this parameter was particularly effective in the low-ovulation-rate group. These results demonstrate that melatonin treatment in the autumn improves embryo quality in aged ewes, and that this effect is not explained by a differential endometrial sensitivity to progesterone. Table 1.Embryo production (mean±s.e.m.) in melatonin-treated (M) and control (C) ewes after superovulation in autumn, and staining intensity of progesterone receptors in the endometrium (CL=corpora lutea)

Autologous mitochondrial transfer as a complementary technique to intracytoplasmic sperm injection to improve embryo quality in patients undergoing in vitro fertilization—a randomized pilot study

To study if autologous mitochondrial transfer (AUGMENT) improves outcome in patients with previously failed invitro fertilization (IVF). Randomized, controlled, triple-blind, experimental study. Private infertility center, Valencian Institute of Infertility (IVI-RMA), Valencia, Spain. Infertile women ≤42years of age, body mass index <30kg/m2, antimüllerian hormone ≥4 pmol/L, >5 million/mL motile sperm, at least one previous IVF with at least five metaphase oocytes (MIIs) collected, and low embryo quality. An ovarian cortex biopsy was performed to isolate egg precursor cells to obtain their mitochondria. Sibling MIIs were randomly allocated to AUGMENT (experimental) or intracytoplasmic sperm injection (Control). In AUGMENT, mitochondrial suspension was injected along with the sperm. Viable blastocysts from both groups were biopsied for preimplantation genetic testing for aneuploidy. Pregnancy, embryo quality. An interim analysis was conducted. The patients' mean age was 36.3 ± 3.6years, and they had an average of 2.5 ± 1.5 previous IVF cycles. Two of the 59 enrolled patients spontaneously conceived (one miscarried). Fifty-seven patients had ovarian biopsies and underwent stimulation. Oocyte retrieval was performed in 56 patients (premature ovulation; n = 1). A total of 253 MIIs were inseminated in AUGMENT and 250 in Control; fertilization rates were 62.7 ± 30.0% and 68.7 ± 29.1%, respectively. Statistical differences were observed in day 5 blastocyst formation rates (23.3 ± 32.0% vs. 41.1 ± 36.9%). Neither the euploid rate per biopsied blastocyst (43.8 ± 41.7% vs. 63.8 ± 44.1%) nor the euploid rate per MII (9.8 ± 20.5% vs. 11.9 ± 16.1%) between AUGMENT and Control achieved statistical significance. Moreover, no differences were seen regarding mitochondrial DNA content and relevant morphokinetic variables. Thirty patients were able to undergo embryo transfer. Cumulative live birth rates per transferred embryo were 41.6% in AUGMENT and 41.2% in Control. AUGMENT does not seem to improve prognosis in this population. Therefore, the study has been discontinued. NCT02586298.

Can peri-ovulatory putrescine supplementation improve egg quality in older infertile women?

The aging-related decline in fertility is an increasingly pressing medical and economic issue in modern society where women are delaying family building. Increasingly sophisticated, costly, and often increasingly invasive, assisted reproductive clinical protocols and laboratory technologies (ART) have helped many older women achieve their reproductive goals. Current ART procedures have not been able to address the fundamental problem of oocyte aging, the increased rate of egg aneuploidy, and the decline of developmental potential of the eggs. Oocyte maturation, which is triggered by luteinizing hormone (LH) in vivo or by injection of human chorionic gonadotropin (hCG) in an in vitro fertilization (IVF) clinic, is the critical stage at which the majority of egg aneuploidies arise and when much of an egg's developmental potential is established. Our proposed strategy focuses on improving egg quality in older women by restoring a robust oocyte maturation process. We have identified putrescine deficiency as one of the causes of poor egg quality in an aged mouse model. Putrescine is a biogenic polyamine naturally produced in peri-ovulatory ovaries. Peri-ovulatory putrescine supplementation has reduced egg aneuploidy, improved embryo quality, and reduced miscarriage rates in aged mice. In this paper, we review the literature on putrescine, its occurrence and physiology in living organisms, and its unique role in oocyte maturation. Preliminary human data demonstrates that there is a maternal aging-related deficiency in ovarian ornithine decarboxylase (ODC), the enzyme responsible for putrescine production. We argue that peri-ovulatory putrescine supplementation holds great promise as a natural and effective therapy for infertility in women of advanced maternal age, applicable in natural conception and in combination with current ART therapies.

Six-week pretreatment with growth hormone improves clinical outcomes of poor ovarian responders undergoing in vitro fertilization treatment: A self-controlled clinical study.

The purpose was to explore whether the 6 weeks of growth hormone (GH) pretreatment could increase the live birth rate of poor ovarian responders (POR). This self-controlled, retrospective study was performed among 380 POR who had GH adjuvant (GH+) at a university-affiliated hospital in Guangzhou, China, from October 2010 to April 2016. Growth hormone was injected daily beginning with the previous menstruation and maintained until ovum pickup, for approximately 6 weeks. Clinical variables between the GH+ cycle and the other GH-free (GH-) cycle of each patient were compared. Both cycles were conducted with a similar conventional control ovarian hyperstimulation protocol for in vitro fertilization treatment. One to one case-control matching was performed to adjust essential confounding factors between GH+ cycles and GH- cycles. GH pretreatment improved embryo quality (1.14 ± 1.50 vs 0.11 ± 0.48, P < 0.05) and decreased miscarriage (18.8% vs 80.0%, P < 0.05) significantly, resulting in an increase in the live birth rate (23.5% vs 3.9%, P < 0.05). The oocyte utilization rate in GH+ cycles was remarkably improved, even with older patients and more failed previous attempts. Significant improvement in embryo quality was shown by an increased number of good-quality embryos and improved oocyte utilization rate after matching. The longer term use of low-dose GH administration for 6 weeks could be beneficial for the utilization of oocytes and for finally increasing the live birth rates of POR.

Effect of the Air Filtration System Replacement on Embryo Quality in the Assisted Reproduction Laboratory.

Improving infrastructural conditions of the in vitro fertilization laboratory, such as the air quality, has profound positive effects on embryo culture. Poor environmental conditions reduce the rate of embryo formation and, therefore, of pregnancy. This review article presents important publications regarding the impact of air quality in human reproduction laboratories on embryo quality, pregnancy success, and live births. The studies demonstrate that the replacing the air filtration system improves significantly the environmental air quality, and, consequently, improves laboratory parameters, such as the fertilization rate, the number of blastocysts, the embryo implantation rate, and the number of live births. On the other hand, improving air quality decreases the number of abortions. Therefore, environmental parameters that improve embryo quality and increase healthy child birth rates must be the main targets for the assisted reproduction laboratory quality control.

Development of bovine embryos in vitro in coculture with murine mesenchymal stem cells and embryonic fibroblasts

Despite the progress on development of new culture media, in vitro-produced embryos still display lower quality when compared to the in vivo-produced counterparts. Coculture has been reconsidered as an alternative to improve embryo quality. Mesenchymal stem cells (MSC) and murine embryonic fibroblasts (MEF) have been extensively used as feeder layers due to their capacity to release growth factors. In the present study we investigated the effect of these feeder layers in oocyte maturation and/or embryo development under in vitro conditions. Oocytes were matured in control (CTRL) conditions or in coculture with MSC or MEF. In vitro fertilization and embryo culture until fourth day were performed in CTRL condition for all groups. Embryos from fourth day on were then cultured until the eighth day in CTRL or in coculture system. No significant differences for metaphase II stage and apoptosis in oocytes were found among the groups. There was also no difference among the groups when we evaluated blastocyst formation on the seventh and eighth day, with exception of a higher hatched blastocyst rate in the group maturated and cultivated in CTRL condition when compared to the group matured and cocultured with MSC. Also no difference was observed in the number of cells in the whole embryos, in the inner cell mass, in the trophoblast and at apoptotic stage on the eighth day. We conclude that coculture with MSC or MEF during maturation and/or embryo development do not enhance the in vitro production of bovine embryos.

Supplementation of IVF solutions with melatonin improves assisted reproductive technology results

Aim: To evaluate the effects of melatonin supplementation of culture medium on the sperm survival rate, fertilization rate, cleavage rate, optimal embryo rate, blastocyst rate, implantation rate and clinical pregnancy rate. Material and Methods: Oocytes and sperm obtained from each patient (86 patients in total) were divided on average into four groups with different concentrations of melatonin (0 nmol/L, 0.1 nmol/L, 1.0 nmol/L, and 10.0 nmol/L). Melatonin was supplemented three times a day to maintain the concentration in different groups, and all the results were recorded at each related time. Results: The results indicated that melatonin could increase sperm survival rate, fertilization rate, and cleavage rate, and the solutions with 10 nmol/L melatonin were most effective (p p Conclusions: Addition of 1.0 nmol/L melatonin into culture media was the most effective to improve embryo quality and clinical outcome of IVF.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P &gt; 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P &lt; 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P &lt; 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P &lt; 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P &gt; 0.05). There was no significant difference (P &gt; 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P &lt; 0.05) and the 1 mg mL−1 group (47.8%; P &lt; 0.05; and 82.6%; P &lt; 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

Relief of endoplasmic reticulum stress enhances DNA damage repair and improves development of pre-implantation embryos.

Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produce better quality embryos compared to late-cleaving embryos. To investigate the significance of endoplasmic reticulum (ER) stress on early embryo cleavage kinetics and development, porcine embryos produced in vitro were separated into early- and late-cleaving groups and then cultured in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Developing embryos were collected at days 3 to 7 of culture for assessment of ER stress status, incidence of DNA double-strand breaks (DSBs), development and total cell number. In the absence of TUDCA treatment, late-cleaving embryos exhibited ER stress, higher incidence of DNA DSBs, as well as reductions in development to the blastocyst stage and total embryo cell numbers. Treatment of late-cleaving embryos with TUDCA mitigated these effects and markedly improved embryo quality and development. These results demonstrate the importance of stress coping responses in early developing embryos, and that reduction of ER stress is a potential means to improve embryo quality and developmental competence.

Frontiers in clinical andrology

Since the introduction of intracytoplasmic sperm injection (ICSI) in 1992 (1) the research into the clinical management of infertile men slowed to a grind in the last three decades (2). Attention has been drawn to assisted reproductive technology (ART) in improving embryo quality and pregnancy outcome. The enthusiasm in sperm function tests, including post coital and sperm penetration tests, in 1990s disappeared and none of these tests have been widely studied clinically. Despite all the pitfalls, conventional semen analysis has remained as one-and-only cornerstone in the evaluation of infertile men over the years (3). The increasing use of ICSI in bypassing severe male factors renders semen analysis a screening tool merely for the presence or absence of sperm. Despite its widespread use, the live birth rate utilizing ICSI as the treatment of male factor infertility is around 30% only (4). This clearly illustrates the pivotal role of paternal contribution to fertilization and development of healthy offspring. A comprehensive evaluation of infertile men and correction of male factors represent the way to improve both natural pregnancy and ART outcomes.

Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk.

Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by in vivo and in vitro oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between in vivo and in vitro oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when in vivo and in vitro EVs were compared (P < 0.01). Interestingly, 97 were exclusively expressed in in vivo EVs, 47 were present only in in vitro and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in in vitro EVs. Moreover, we showed that in vitro-produced embryos were able to internalize in vivo EVs during culture with a functional effect in the embryo development. In vivo EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under in vitro conditions.

Effects of dry matter and energy intake on quality of oocytes and embryos in ruminants.

The success of herd fertility involves the development of healthy follicles, viable oocytes and embryos capable of establishing and maintaining a pregnancy. Herein we discuss how nutrition interacts with reproduction throughout follicle development and pregnancy establishment, focusing on dry matter and energy intake. High feed intake, especially associated with moderate to high body condition, before and through superstimulation protocols, natural or induced single-ovulations or before ovum pick-up has detrimental effects on the quality of oocytes or embryos. Feed restriction or high energy supply can be used strategically to obtain either more or better quality oocytes or embryos. Altering diets that provide different concentrations of circulating insulin may improve ovarian status, oocyte quality, embryo development and pregnancy establishment and maintenance. Some sources of fat can positively affect reproductive performance, such as polyunsaturated fatty acids, improving embryo quality and pregnancy. In contrast, fat supplementation in the diet may compromise embryo cryotolerance. Finally, nutrition can alter concentrations of circulating or intrafollicular hormones and metabolites and the expression of genes in cattle oocytes and embryos. For an adequate feeding program to benefit reproductive performance, factors such as genetic group, source of energy, metabolic status, physiological status and level of feed intake must be taken into account.

The embryonic stress response to in vitro culture: insight from genomic analysis.

Recent genomic studies have shed light on the impact of in vitro culture (IVC) on embryonic homeostasis and the differential gene expression profiles associated with lower developmental competence. Consistently, the embryonic stress responses to IVC conditions correlate with transcriptomic changes in pathways related to energetic metabolism, extracellular matrix remodelling and inflammatory signalling. These changes appear to result from a developmental adaptation that enhances a Warburg-like effect known to occur naturally during blastulation. First discovered in cancer cells, the Warburg effect (increased glycolysis under aerobic conditions) is thought to result from mitochondrial dysfunction. In the case of IVC embryos, culture conditions may interfere with mitochondrial maturation and oxidative phosphorylation, forcing cells to rely on glycolysis in order to maintain energetic homeostasis. While beneficial in the short term, such adaptations may lead to epigenetic changes with potential long-term effects on implantation, foetal growth and post-natal health. We conclude that lessening the detrimental effects of IVC on mitochondrial activity would lead to significantly improved embryo quality.

Expression of Na+/H+ exchanger isoforms 1, 2, 3, and 4 in bovine endometrium and the influence of uterine pH at time of fixed-time AI of pregnancy success

Cows that exhibit estrus prior to fixed-time AI had increased sperm transport to the site of fertilization, and improved embryo quality on d 6 after insemination. Sperm transport is influenced by uterine pH, and research has reported that uterine pH decreased at onset of estrus, but must return to normal prior to ovulation. Therefore, the objectives of these studies were to investigate a possible mechanism for the regulation of uterine pH around the onset of estrus, and to determine if uterine pH at time of fixed-time AI influenced pregnancy success. In experiment 1, Angus-cross beef cows (n=40 and 28 in rep. 1 and 2, respectively) were synchronized with the PG 6-day CIDR protocol (PGF2α on d -9, GnRH and insertion of a CIDR on d -6, and PGF2α and CIDR removal on d 0). Cows were blocked by follicle size at time of CIDR removal, and uterine biopsies were collected at 0, 12, 24, 36, 48, 60 (Rep. 1), 72, 84, or 96h (Rep2) after CIDR removal, and total cellular RNA was extracted from all biopsies. Estrus was monitored by the HeatWatch Estrous Detection System. In experiment 2, 223 postpartum beef cows in 2 herds were synchronized with a fixed-time AI protocol (herd 1: n=97; CO-Synch plus CIDR protocol; herd 2: n=126; Co-synch protocol). Uterine pH was determined at time of AI (n=80 and 63 for herd 1 and 2, respectively), and estrus was monitored by visual estrus detection with the aid of an ESTROTECT estrous detection patches, and pregnancy was determined by transrectal ultrasonography. In experiment 1, there was a significant (P<0.01), quadratic relationship in expression of Na+/H+ exchanger isoforms 1, 2, and 3 among animals that exhibited estrus, with expression greatest at time of CIDR removal, decreasing to the onset of estrus, and then increasing again following the onset of estrus. Among cows that did not exhibit estrus, the preceding relationship did not exist (P>0.46). In experiment 2, cows that had initiated estrus prior to fixed-time AI had decreased (P=0.01) uterine pH compared to cows that did not initiate estrus (6.78±0.03 and 6.89±0.03, respectively), and uterine pH at AI had an approximately linear effect on pregnancy success within the observed pH range. Furthermore, cows that initiated estrus prior to AI had increased (P=0.05) pregnancy success (52% vs. 38%) compared to cows that had not initiated estrus. In summary, expression of Na+/H+ exchanger isoforms 1, 2, and 3 decreased after CIDR removal among cows that exhibited estrus, but did not change among cows that did not exhibit estrus. Additionally, as uterine pH decreased pregnancy success tended to increase (P=0.076, logistics regression). Thus, Na+/H+ exchanger isoforms 1, 2, and 3 appear to be key regulators of uterine pH around the onset of estrus, and this change in uterine pH is critical for pregnancy success. Summary sentenceExpression of Na+/H+ exchanger isoforms 1, 2, and 3 decreased after CIDR removal among cows that exhibited estrus, but did not change among cows that did not exhibit estrus, and as uterine pH decreased, pregnancy success tended to increase.

Superovulation in wood bison (Bison bison athabascae): Effects of progesterone, treatment protocol and gonadotropin preparations for the induction of ovulation

Experiments were done to determine the ovarian response and embryo production following superstimulation of wood bison. In Experiment 1 (Anovulatory season), the efficacy of pLH vs. hCG for inducing ovulation was compared in wood bison superstimulated with a single dose of pFSH in 0.5% hyaluronan and the effect of exogenous progesterone (PRID) on superovulatory response and embryo quality was examined. In Experiment 2 (Ovulatory season), the efficacy of pLH vs. hCG for the induction of ovulation was compared in wood bison superstimulated with pFSH in a single intramuscular dose vs. a two-dose regimen 48h apart (split dose) in 0.5% hyaluronan. In Experiment 1, the number of CL was greater (P<0.05) in bison treated with hCG than pLH (6.6±1.8 vs. 2.8±0.8) and in those that were not given PRID (6.0±1.5 vs. 2.7±1.0). There was no effect of progesterone treatment on embryo quality. In Experiment 2, the number of CL was greater (P<0.05) in bison treated with hCG than with pLH (6.3±0.8 vs. 3.8±1.2) and in bison superstimulated with split dose vs. single dose of FSH (7.1±0.9 vs. 3.0±0.8). The number of ova/embryos and freezable embryos did not differ among groups in either experiment. In conclusion, hCG induced a greater ovulatory response than pLH in both seasons. Two doses of FSH induced the greatest superovulatory response during the ovulatory season. Exogenous progesterone did not improve embryo quality during the anovulatory season.

220 EFFECT OF ZINC SUPPLEMENTATION DURING IN VITRO MATURATION ON EQUINE BLASTOCYST RATES AFTER INTRACYTOPLASMIC SPERM INJECTION

Use of intracytoplasmic sperm injection (ICSI) in horses has growing clinical and research importance; however, little is known of factors affecting efficacy of this system. Supplementation of zinc during in vitro maturation (IVM) has been shown to increase oocyte glutathione levels, decrease reactive oxygen species, and increase blastocyst rates in vitro in cattle and pigs, but has not been evaluated in the horse. In this study, we examined the effect of zinc supplementation during IVM on rates of maturation and blastocyst formation after ICSI. Oocytes were collected from follicles ≥5 mm in diameter in live mares. Blood serum and follicular fluid from 15- to 30-mm follicles were collected from 3 mares for zinc analysis by flame atomic absorption spectrometry. Oocytes were held overnight at room temperature (Choi et al. 2006 Theriogenology 66, 955–963), and then randomly assigned to IVM in the presence of 1 of 4 concentrations of added zinc (0.0, 0.5, 1.0, and 1.5 μg mL–1), added as ZnSO4·7H2O. The oocytes were cultured for IVM in M199 with Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum (FBS) for 30 h. The oocytes were then denuded of cumulus and those with a polar body subjected to ICSI with frozen-thawed sperm. Presumptive zygotes were cultured in a commercial human embryo culture medium (LifeGlobal; http://www.lifeglobalgroup.com/), supplemented with 10% FBS, under 6% CO2, 5% O2, and 89% N2 at 38.2°C. On Day 5 (Day 0 = injection day), embryos were evaluated for presumptive cleavage and transferred to medium with 20 mM added glucose. Blastocyst formation was evaluated on Days 7 to 11. Data were analysed by Fisher’s exact test. The mean zinc concentrations in mare serum and follicular fluid were 0.50 and 0.44 μg mL–1, respectively. The mean zinc concentration of the FBS was 2.70 μg mL–1, and that of IVM medium containing 10% FBS were 0.28, 0.80, 1.23, and 1.68 μg mL–1 for the 0.0, 0.5, 1.0, and 1.5 μg mL–1 added-zinc treatments, respectively. The rates of oocyte maturation were not significantly different among treatments (43/61, 70%; 46/70, 66%; 45/71, 63%; and 49/70, 70%, respectively). Neither cleavage rates (81–92%) nor blastocyst rates (12/42, 29%; 9/43, 21%, 8/44, 18%, and 15/48, 31%, respectively) differed significantly among treatments. However, the proportion of blastocysts that developed on Day 7 out of total blastocysts was higher for the combined added-zinc treatments (0.5, 1, and 1.5 μg mL–1) than for the treatment with no added zinc (15/32, 47% v. 1/12, 8%, respectively; P &lt; 0.05). These results indicate that supplementation of zinc to the IVM medium used did not influence equine oocyte maturation or blastocyst development rates but may have improved embryo quality, as reflected in earlier blastocyst development. The more subtle response seen, compared with that reported in other species, may be because the 10% FBS resulted in zinc concentrations in the basal medium (0.28 μg mL–1) only slightly lower than that in equine follicular fluid (0.44 μg mL–1). This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University, and by the Clinical Equine ICSI Program, Texas A&amp;M University.

L-ergothioneine supplementation during culture improves quality of bovine in vitro–produced embryos

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro–produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%–40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.

Early limited exposure to a microfluidic environment appears to enhance embryo development in vitro

The push toward single embryo transfer (SET), has created an ever increasing need for high quality embryos. Numerous groups have proposed the use of microfluidics (MF) as a means of increasing embryo quality. However, to date, no practical means of creating a MF environment (MFE) has been developed. This laboratory has previously reported on the use of a Modified Specific Gravity Technique (MSGT) in determining the best embryos for transfer. Results from that study suggested a potential improvement in embryo quality due to a momentary (mins) exposure to a MFE.