Production Of Viable Embryos Research Articles

Maternal regulation of the vertebrate oocyte-to-embryo transition.

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.

P-681 LH priming before ovarian stimulation in POSEIDON-4 patients: effects on oocyte recovery and post ICSI/IVF pregnancy rates

Abstract Study question Can a seven-day long LH priming treatment preceding ovarian stimulation (OS) improve follicular responses, oocyte yields and ICSI/IVF outcomes of POSEIDON-4 patients? Summary answer A seven-day long LH priming can substantially increase pregnancy rates through specific benefits in different subgroups of POSEIDON-4 patients. What is known already The combination of advanced maternal age (AMA) with diminished ovarian reserve (DOR) has become one of the most relevant and complex topics in the field of reproductive medicine. AMA/DOR patients appear to be refractory to increased gonadotropin dose and may benefit from strategies specifically capable of increasing the population of FSH-responsive small follicles before the onset of ovarian stimulation (OS). Androgen mediated LH intra-ovarian activity stimulates early follicular development. Pre-treatment with LH has been suggested to improve ICSI/IVF outcomes in normal responders but has not been assessed in AMA/DOR patients, despite the prevalence and relevance of this population. Study design, size, duration Retrospective cohort study from 2022 to 2023, including 235 POSEIDON-4 patients stimulated with FSH+LH in a flexible antagonist protocol with oestradiol pre-treatment (n = 129) or with the same stimulatory treatment preceded by a seven-day LH priming (150 IU/day) combined with GnRH agonist downregulation (n = 106). Follicular responses, oocyte recovery and ICSI/IVF outcomes were compared between treatment-groups in overall POSEIDON-4 patients and in a subgroup of patients with AMH <0.75 ng/mL (n = 77/Control, n = 59/LH priming). Participants/materials, setting, methods All participants were POSEIDON-4 caucasian patients aged 35 to 44 years undergoing a single homologous ICSI/IVF cycle in our clinic. Outcomes in the form of percentages were compared with the Fisher’s exact test, whereas differences in continuous variables were assessed with the Wilcoxon sum rank test. A multivariate analysis was performed to control for the interference of unequal variations in maternal age, AMH, insemination method and presence of male subfertility. Main results and the role of chance Patients in different treatment groups did not differ (p > 0.05) regarding maternal age (39.2±2.5 vs. 39.3±2.5), basal FSH serum concentrations (11.0±4.6 vs. 10.1±4.3) and antral follicle count (6.3±3.0 vs. 6.4±3.3, for patients stimulated with and without LH priming, respectively), but patients treated with LH priming presented slightly lower AMH serum levels (0.56±0.3 vs. 0.63±0.3; p = 0.05). Nevertheless, patients treated with LH priming achieved a twice higher clinical pregnancy rate per cycle (19.8% vs. 9.3%; p = 0.02), accompanied by a 40% reduction in cycle cancellation (32.1% vs. 45.0%; p = 0.03) and increased production of viable embryos (transferred fresh + frozen) per cycle (1.22±1.09 vs. 0.94±1.07; p = 0.02) and per oocyte inseminated (48.3% vs. 38.5%; p = 0.01), but did not differ regarding follicular outputs or oocyte yield. The multivariate analysis indicated a robust association between LH priming and pregnancy achievement, independently of potential confounding variables (OR = 2.52; p = 0.02). A two-fold increase in pregnancy achievement was also observed in severely restricted POSEIDON 4 patients (AMH <0.75 ng/mL) subjected to the LH priming (p = 0.05), which in this case was accompanied by higher oocyte yields per cycle (3.3±2.8 vs. 2.2±2.0; p = 0.04) and per pick (3.9±2.6 vs. 2.3±2.0; p < 0.01). Limitations, reasons for caution This study is limited by its retrospective nature and the data may be potentially influenced by the use of different downregulation strategies in the treatment-groups, even if the literature indicates equivalent performance for these strategies in the population under analysis. Wider implications of the findings The present data strongly suggest that a seven-day LH priming preceding OS can markedly improve ICSI/IVF outcomes of POSEIDON-4 patients, while indicating that this strategy benefits in different ways subgroups of this increasingly prevalent patient category. These findings thus represent new valuable parameters for the progress of ovarian stimulation. Trial registration number NA

P-730 Detection of mitochondrial reversal following meiotic spindle transfer: a finding of importance for mitochondrial replacement therapies used for the purpose of avoiding mtDNA disease transmission

Abstract Study question Following transfer of the meiotic spindle from a patient’s oocyte into an enucleated donor oocyte, do relative levels of patient and donor mtDNA remain stable? Summary answer mtDNA transferred along with the patient’s spindle typically remain at a low level. However, disproportionate expansion of the patient’s mtDNA can occur in some cases. What is known already Mitochondrial DNA disorders are caused by mutations in the mitochondrial genome, disrupting ATP production. They have few treatment options and no cure. All mitochondria are derived from the oocyte, therefore mtDNA disorders are maternally inherited. It has been proposed that disease transmission could be avoided if female mtDNA mutation carriers underwent meiotic spindle transfer (MST), removing the chromosomes (on the spindle) from an affected oocyte and transferring them into the healthy cytoplasm of a donor oocyte. However, research using embryonic stem cells has suggested that the small population of mitochondria transferred along with the spindle can sometimes undergo dramatic expansion. Study design, size, duration 25 infertile couples were enrolled in a prospective pilot study evaluating MST as a treatment for infertility. The female participants had a mean age of 37.2, and an average of 6.4 previous unsuccessful IVF cycles (range 3-11) characterised by extremely poor embryo development and without any previous pregnancy. Importantly, none of the patients were carriers of a mtDNA disorder. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. Participants/materials, setting, methods Metaphase-II-spindles from patient oocytes were transferred into enucleated donor oocytes. Reconstructed oocytes underwent ICSI and the resulting embryos were transferred at the blastocyst stage. Mitochondrial genomes were sequenced to identify polymorphisms differing between the patient and oocyte donor. These variations were quantified in the embryos (blastocyst biopsies), during pregnancy (amniocentesis), in newborns (cord blood, cord tissue, urine), at 3-6 months and at one year (saliva, urine, blood), revealing the relative amounts of each mitochondrial type. Main results and the role of chance This MST pilot study resulted in the birth of six children, indicating that the procedure is compatible with the production of viable embryos, capable of producing healthy live births. The patient’s mtDNA was shown to represent <1% of the total in all blastocysts produced, confirming that MST is highly reproducible and that relatively few mitochondria are transferred along with the spindle. For five of the six children, the proportion of the total mtDNA attributable to the patient appeared to be stable, remaining at very low levels in all of the samples from later developmental stages. However, in one child the small quantity of mtDNA transferred along with the spindle increased disproportionately with respect to the mtDNA of the oocyte donor, ultimately representing 30-60% of the total at birth, depending on the tissue tested. The precise timing of the expansion of one type of mtDNA at the expense of the other is not known but occurred sometime between the blastocyst stage and birth. By the time of birth, the levels of donor and patient mtDNA appeared to have stabilised (no further increases were seen at 6 months and one year). All of the children born remain developmentally normal and healthy. Limitations, reasons for caution After using MST, several pregnancies were achieved for patients with a long history of unsuccessful IVF attempts, associated with poor oocyte quality and a failure to produce blastocysts. However, this small pilot study lacked the controls necessary for a definitive evaluation of MST as a tool for infertility treatment. Wider implications of the findings All children born following MST were healthy. However, our results clearly demonstrate that a substantial degree of mtDNA ‘reversal’ is possible. Consequently, mitochondrial replacement therapies used for avoidance of mtDNA disorders might not always be successful, even when initial levels of mutant mtDNA in reconstructed oocytes are very low. Trial registration number ISRCTN11455145

Study of the factors affecting embryo yields and quality in superovulated Morada Nova ewes that underwent non-surgical uterine flushing.

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9SOV , n=21) or 12-day (G-12SOV , n=21) progesterone (P4 )-based estruses synchronization protocol coupled with superovulatory treatment with decreasing doses of porcine follicle-stimulating hormone (133mg of pFSH given over 3days). Non-surgical embryo recovery (NSER) was performed 6-7days after the onset of oestrus. Total antral follicle count doubled from the first to the sixth pFSH dose in both groups (p<.05). Oestrus responses did not vary between the two groups of animals (95.2%). Corpora lutea (CL) were detected in 85.0% and 60.0% of ewes that previously manifested oestrus behaviour in G-9SOV and G-12SOV respectively. NSER was successfully completed in 86.2% of ewes that had CL (p>.05). The mean number of CL per ewe/successfully flushed donor ewe was greater (p<.05) in G-12SOV (12.3±1.7/12.1±1.9) than in G-9SOV (7.9±1.4/8.2±1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p>.05) in G-12SOV (5.8±1.9 and 3.7±1.7) than G-9SOV (3.5±1.1 and 0.8±0.3 respectively). The total follicle count (all follicles ≥2mm in diameter) at the sixth pFSH dose (at P4 -device removal) was positively correlated (p<.05) with the number of CL (r=.95) and viable embryos (r=.91) in G-12SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12SOV group had better superovulatory responses compared with G-9SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P4 for 12days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes.

In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos

ABSTRACT In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P &lt; 0.0001; n = 681 vs. 509 for grade 2 COC, P = 0.010, for zebu and taurine donors, respectively). There were differences in embryo production percentages obtained from OM (0.44% from zebu and 6.42% from taurine, P = 0.017), BL (14.18% from zebu and 3.74% from taurine, P &lt; 0.0001), and BX (81.43% from zebu and 75.13% from taurine, P &lt; 0.0001). No significant difference was observed for embryo production from BI and pregnancy rate (P &gt; 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.

Follicular dynamics and in vivo embryo production in Santa Inês ewes treated with smaller doses of pFSH

To evaluate follicular dynamics, there was assessment of superovulatory response and in vivo embryo production in ewes treated with relatively smaller doses of exogenous pFSH than typically used in combination with a dose of eCG at the beginning of the gonadotropin treatment period. Santa Inês ewes (n = 24) were randomly divided into three groups, based on mg dose of pFSH administered: G200 (n = 8), G133 (n = 8) and G100 (n = 8) in eight decreasing doses at 12 -h intervals. All ewes were treated with 300 IU of eCG concomitantly starting with first pFSH administration. Ovulatory follicular dynamics and follicular wall vascularization (FWV) were evaluated using a B-mode and color Doppler ultrasonic machine, respectively. Superovulatory response and embryo production were evaluated 6 days after estrous detection. In the G200 group, the preovulatory follicle size (PFS) were less (P < 0.05), ovulation time later (P < 0.05), and PFS rate greater (P < 0.05); while in the G100 group ovulation rate, and number and percentage of unfertilized eggs were greater (P < 0.05) than in the G133 group (P < 0.05). Number and percentage of viable embryos were greater in the G200 and G100 compared to G133 group (P < 0.05). The dose of 100 mg of FSH was as efficacious as the traditional dose of 200 mg, in combination with a dose of eCG, for superovulatory response and viable embryo production but there was a greater percentage of unfertilized eggs with this treatment.

Enzymatic activity of mouse group X-sPLA2 improves in vitro production of preimplantation bovine embryos

Assisted reproductive technologies (ART) are widely used for both humans and domestic animals. In bovine species, in vitro embryo production is increasingly used and significant efforts are being made to optimize media and culture conditions. Phospholipase A2 (PLA2) are lipolytic enzymes that hydrolyze glycerophospholipids to produce free fatty acids and lysophospholipids that have been found to be critical for many biological processes. Mouse group X secreted PLA2 (mGX) is abundant in the male reproductive tract and its use during sperm capacitation has been shown to improve in vitro production of viable embryos in a mouse model. Here, we examined its effect in the bovine species, testing the impact of mGX on the three steps involved in vitro production of preimplantation embryos: oocyte maturation, fertilization and preimplantation development. We found that incubating cumulus oocyte complexes (COC) or gametes with mGX resulted in increased blastocyst hatching and blastocyst production, respectively. The increases of embryo production induced by the phospholipase mGX were not observed for the catalytically inactive mutant H48Q-mGX, suggesting that these effects require the enzymatic activity of mGX. We also tested bGIB, a bovine homolog of mGX. bGIB failed to improve blastocyst production, underlining the high specificity of mGX. In conclusion, the results presented show that the effects of mGX are not restricted to the mouse model and that it is potent in the bovine species as well. This result strengthens the potential of mGX as a “pro-fertility drug” for mammalian reproduction.

Zinc availability during germline development impacts embryo viability in Caenorhabditis elegans

Zinc is an essential metal that serves as a cofactor in a variety of cellular processes, including meiotic maturation. Cellular control of zinc uptake, availability and efflux is closely linked to meiotic progression in rodent and primate reproduction where large fluctuations in zinc levels are critical at several steps in the oocyte-to-embryo transition. Despite these well-documented roles of zinc fluxes during meiosis, only a few of the genes encoding key zinc receptors, membrane-spanning transporters, and downstream signaling pathway factors have been identified to date. Furthermore, little is known about analogous roles for zinc fluxes in the context of a whole organism. Here, we evaluate whether zinc availability regulates germline development and oocyte viability in the nematode Caenorhabditis elegans, an experimentally flexible model organism. We find that similar to mammals, mild zinc limitation in C. elegans profoundly impacts the reproductive axis: the brood size is significantly reduced under conditions of zinc limitation where other physiological functions are not perturbed. Zinc limitation in this organism has a more pronounced impact on oocytes than sperm and this leads to the decrease in viable embryo production. Moreover, acute zinc limitation of isolated zygotes prevents extrusion of the second polar body during meiosis and leads to aneuploid embryos. Thus, the zinc-dependent steps in C. elegans gametogenesis roughly parallel those described in meiotic-to-mitotic transitions in mammals.

Stress response to anthropogenic noise in Atlantic cod Gadus morhua L.

The potential effects of anthropogenic noise on the physiology of Atlantic cod have not been well described. The aim of the present study was to investigate the impact of anthropogenic noise on Atlantic cod stress response using cortisol as a biomarker as well as on broodstock spawning performance. Results showed that artificial noise consisting of a linear sweep from 100 to 1000Hz can induce a transient and mild cortisol elevation with a clear noise intensity dose response. In all cases plasma levels returned to baseline levels <1h post sound exposure. Daily exposure to a similar intensity and frequency noise range applied habitually to a broodstock population during the spawning window resulted in a significant reduction in total egg production and fertilisation rates thus reducing the total production of viable embryos by over 50%. In addition, a significant negative correlation between egg cortisol content and fertilisation rate was observed. These results confirm that cod can perceive noise generated within a frequency range of 100–1000Hz and display a heightened cortisol plasma level. In addition, anthropogenic noise can have negative impacts on cod spawning performances.

Physiological management of dietary deficiency in n-3 fatty acids by spawning Gulf killifish (Fundulus grandis)

Lipid dynamics of spawning fish are critical to the production of viable embryos and larvae. The present study utilized manipulation of dietary fatty acid (FA) profiles to examine the ability of spawning Gulf killifish (Fundulus grandis) to mobilize critical lipid components from somatic reserves or synthesize long-chain polyunsaturated FAs (LC-PUFAs) de novo from shorter-chain C18 precursors. An egg and multi-tissue evaluation of changes in FA concentrations across time after fish were switched from LC-PUFA-rich to LC-PUFA-deficient experimental diets was employed. The two experimental diets contained lipid sources which differed drastically in n-3 C18 FA content but had similar levels of n-6 C18 FAs. Discrete effects of dietary n-3 FAs can be analyzed because n-3 and n-6 represent distinct metabolic families which cannot be exchanged in vivo. Results indicate that a combination of mobilization and de novo synthesis is likely utilized to maintain physiologically required FA levels in critical tissues and embryos. Mobilization was supported by decreases in LC-PUFAs in somatic tissues and decreases in intraperitoneal fat content and liver mass. Evidence for biosynthesis was provided by a higher level of n-3 LC-PUFAs in the liver and ova of fish fed diets containing n-3 C18 precursors versus those fed diets with low levels of precursor FAs. The characteristic physiological plasticity of Gulf killifish is exemplified in the nutritional domain by its management of dietary FA deficiency.

Rapid and Efficient Identification of Caenorhabditis elegans Legacy Mutations Using Hawaiian SNP-Based Mapping and Whole-Genome Sequencing.

The production of viable embryos requires the coordination of many cellular processes, including protein synthesis, cytoskeletal reorganization, establishment of polarity, cell migration, cell division, and in Caenorhabditis elegans, eggshell formation. Defects in any of these processes can lead to embryonic lethality. We examined six temperature-sensitive mutants as well as one nonconditional mutant that were previously identified in genetic screens as either embryonic lethal (maternal-effect or zygotic lethal) or eggshell defective. The responsible molecular lesion for each had never been determined. After confirmation of temperature sensitivity and lethality, we performed whole-genome sequencing using a single-nucleotide polymorphism mapping strategy to pinpoint the molecular lesions. Gene candidates were confirmed by RNA interference phenocopy and/or complementation tests and one mutant was further validated by CRISPR (Clustered Regularly Interspaced Short Palidromic Repeats)/Cas9 gene editing. This approach identified new alleles of several genes that had only been previously studied by RNA interference depletion. Our identification of temperature-sensitive alleles for all of these essential genes provides an extremely useful tool for further investigation for the C. elegans community, such as the ability to address mutant phenotypes at various developmental stages and the ability to carry out suppressor/enhancer screens to identify other genes that function in a specific cellular process.

Effect of natural mating or laparoscopic artificial insemination in superovulated Santa Inês ewes on superovulatory response, fertility and embryo viability

This study evaluated the effect of two mating methods (GNM: natural mating or GAI: laparoscopic artificial insemination) on superovulatory response, fertility and embryo yield in superovulated ewes. Fifteen non-pregnant Santa Inês ewes were superovulated and either mated by GNM or GAI in a crossover design. Oestrus was synchronised using intravaginal progestagen sponges for 6 days and on Day 5, 300 IU eCG and 0.0375 mg d-cloprostenol were given. Twelve hours after sponge removal, 0.025 mg gonadotropin-releasing hormone was administered. Superovulation started 48 h after gonadotropin-releasing hormone treatment, using 5 IU/kg follicle-stimulating hormone (pFSH). At the first pFSH dose, new sponges were inserted. At the fifth dose, 0.0375 mg cloprostenol was administered and the sponges were removed. The GNM was mated with rams every 12 h, until the end of oestrus. The ewes of GAI were laparoscopic inseminated with frozen–thawed semen 36 and 48 h after sponge removal. Ultrasonography was performed every 24 h from the beginning of oestrus synchronisation treatment and every 12 h from the second sponge removal to 2 days after the last pFSH dose. Six to seven days after mating, the number of corpora lutea (CL) was evaluated by laparoscopy and the females with &gt; 4 CL were subjected to embryo collection. The interval from sponge removal to ovulation was shorter (P &lt; 0.05) in the GNM. The overall superovulatory response was 63.3% (19/30), with 60.0% and 66.7% in GNM and GAI, respectively (P &gt; 0.05). The number of recovered structures (6.4 ± 2.4 vs 4.5 ± 3.0), recovery rate (74.0 ± 16.0 vs 52.3 ± 26.5%), number of transferable embryos (3.0 ± 2.9 vs 3.6 ± 2.0) and viability rate (47.2 ± 45.3 vs 77.4 ± 37.1%) did not differ between GAI and GNM (P &gt; 0.05). However, the GAI group showed a higher (P &lt; 0.05) number of unfertilised oocytes (3.1 ± 3.1) and a higher non-fertilisation rate (47.1 ± 45.3%) than the GNM (0.9 ± 2.1 and 11.5 ± 21.5%). The mating method did not affect the superovulatory response, and production of viable embryos although the non-fertilisation rate has been inferior for the AI group.

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.

Reproductive technologies and the porcine embryonic transcriptome

The domestic pig is not only an economically-important livestock species, but also an increasingly recognized biomedical animal model due to its physiological similarities with humans. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in the pig using either in vivo or in vitro production methods. The application of assisted reproductive technologies (ART) has the potential to increase reproductive efficiency in livestock. These technologies include, but are not limited to: artificial insemination (AI), fixed-time AI, embryo transfer, cryopreservation of sperm/oocytes/embryos, in vitro fertilization and somatic cell nuclear transfer (cloning). However, the application of ART is much less efficient in the pig than in many other mammalian species such as cattle. Until recently, the underlying causes of these inefficiencies have been difficult to study, but advances in molecular biology techniques for studying gene expression have resulted in the availability of a variety of options for gene expression profiling such as microarrays, and next generation sequencing technologies. Capitalizing on these technologies the effects of various ARTs on the porcine embryonic transcriptome has been determined and the impact on the related biological pathways and functions been evaluated. The implications of these results on the efficiency of ARTs in swine, as well potential consequences for the developing embryo and resulting offspring, are reviewed.

In vivo embryo production in cows superovulated 1 or 2 days after ovum pick-up

The present study evaluated superovulatory responses and in vivo embryo production in cows treated with FSH starting 1 or 2 days after ovum pick-up (OPU). Thirty-three non-lactating Nelore cows were subjected to aspiration of all follicles ≥3mm for OPU. After OPU, cows were randomly divided into two groups in which the follicle superstimulatory treatments with FSH started 1 or 2 days after OPU (Groups D1 and D2, respectively). Data are presented as the least squares mean±s.e.m. The number of follicles ≥3mm before OPU was similar between groups (~34); however, cows in Group D2 had more follicles ≥3mm on the first day of FSH (15.2±2.3 vs 7.6±1.7; P=0.04) and a higher ratio of the number of follicles at first FSH/number of follicles before OPU (0.41±0.04 vs 0.24±0.02; P=0.01). In addition, Group D2 cows had a greater superovulatory response than did cows in Group D1 (18.9±2.8 vs 9.1±1.9 corpora lutea, respectively; P<0.03). However, there was no difference in the total number of recovered ova and embryos from cows in Groups D2 and D1 (5.1±1.4 vs 4.9±1.3, respectively; P>0.10). Nevertheless Group D2 cows had more freezable embryos than Group D1 cows (3.2±1.1 vs 1.3±0.5, respectively; P<0.05). Cows from Group D2 had a much higher proportion (P<0.001) of follicles ≥8mm compared with follicles ≥6mm and <8mm at the time of the last treatment with FSH. In conclusion, to obtain a greater production of viable embryos in superovulated cows after OPU, it is recommended to wait at least 2 days before starting FSH treatment.

Activation of dormant follicles followed by production of viable embryos and offspring via in vitro maturation and fertilization

Activation of dormant follicles followed by production of viable embryos and offspring via in vitro maturation and fertilization

318 EFFICIENCY OF PROTOCOL P-36, ASSOCIATED WITH EQUINE CHORIONIC GONADOTROPIN OR LUTEINIZING HORMONE ADMINISTRATION, IN THE LAST DAY OF SUPERESTIMULATORY TREATMENT, IN NELLORE COWS

The objective of this study was to evaluate the use of pLH in replacement of eCG on the last day of P-36 superstimulatory treatment in Nellore donors. Recent studies demonstrated improvement in embryo production when the last 2 doses of FSH were replaced by eCG. However, consecutive use of eCG in bovine superstimulatory protocols may induce antibody against eCG, decreasing embryo production. Twenty-five Nellore cows were randomly allocated in 4 groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent 4 treatments in a cross-over design. Donors received an intravaginal device containing 1.0 g of progesterone (IVD, Primer®, Tecnopec, Sao Paulo, Brazil) and oestradiol benzoate (2.0 mg, IM; Estrogin®, Farmavet, Sao Paulo, Brazil) at a random stage of the oestrous cycle (D0). Cows from the control group were superestimulated with decreasing doses of pFSH (133 mg, IM; Folltropin-V®, Bioniche, Ontario, Canada; D5-8). In the P-36/eCG group, the last 2 doses of pFSH were replaced by 2 doses of eCG (200 IU each dose, IM; Folligon®, Intervet, Boxmeer, the Netherlands). In the P-36/LH2 and P-36/LH4 groups, the last 2 doses of pFSH were replaced by 2 doses of 1 and 2 mg of pLH, respectively (IM; Lutropin®, Bioniche). All animals were treated with prostaglandin F2α (150 μg d-cloprostenol, IM; Prolise®, Tecnopec) on D7, and the IVD was removed 36 h after. Ovulation was induced with 12.5 mg of pLH (IM) on D9, and all animals received fixed-time artificial insemination 12 and 24 h after pLH. Embryo flushing was performed on D16. Data were analysed by ANOVA (Proc Mixed, SAS). There was a significant difference in the number of corpus luteum in the eCG group (19.2 ± 2.4) when compared with the LH2 (12.7 ± 2.0) and LH4 groups (12.3 ± 1.5; P &lt; 0.05). In addition, there was a tendency of lower ovulation rate in the LH2 group as compared with the eCG group (50.6 and 67.8%, respectively; P = 0.06). However, there was no difference in viable embryo yield among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0); P &gt; 0.05. In conclusion, eCG can be replaced by pLH (4.0 mg), in the last day of P-36 protocol, without affecting the production of viable embryos in Nellore cows. The authors acknowledge FAPESP (Sao Paulo, Brazil) for funding and fellowships for A. C. S. Oliveira, M. C. C. Mattos, and M. R. Bastos.

Embryo and larva development in common dentex ( Dentex dentex), a pelagophil teleost: The quantitative composition of egg-free amino acids and their interrelations

Free amino acids (FAAs) play a key role in the physiology of marine teleosts (eggs, embryos, and larvae). However, the relationship between the egg FAAs content and the production of viable embryos and larvae (at different developmental stages) in batch spawner pelagophils has not yet comprehensively been investigated. Viable eggs of common dentex, Dentex dentex, were obtained from captive broodstocks. Egg wet weight (WW), dry weight (DW), and water content (%W) and viability parameters, or VPs (egg floating rate [FR], hatching rate [HR], and larval survival rate [SR] at days 0 to 5 posthatch) were determined for 45 egg batches. The egg batches were classified according to their HR magnitude. Twelve egg batches with the same WW, DW, and %W were taken from the same broodstock and at the same developmental stage to determine the qualitative and quantitative composition of FAAs. The total FAA (TFAA) content, glutamic acid (Glu), asparagine (Asn), glutamine (Gln), and arginine (Arg) were correlated with VPs. The Glu was significantly correlated with HR and SR at 0 day posthatch (dph), the Asn with SR at 1 dph, and the Gln and Arg with FR and HR. Of the 361 ratios made based on the absolute concentrations of FAAs, 24 ratios were correlated with VPs (P < 0.005) through 42 simple regression models (R 2 = 0.641 to 0.846). Of the 42 significant relationships found ∼10%, ∼28%, ∼12%, ∼30%, ∼8%, ∼4%, ∼2%, ∼2%, and ∼2% of the models show the relations of the egg FAAs ratios with FR, HR, SR at days 1 to 5 posthatch, and %W, respectively. A path coefficient in combination with a Pearson's correlation coefficient provided a series of statistical evidences to show the effects of the egg FAAs interrelations on the relationships found between quantitative composition of a FAA and a VP.

Essential but unhelpful wasp Wolbachia

Essential but unhelpful wasp Wolbachia

137 DIFFERENCES IN EMBRYO PRODUCTION BETWEEN LACTATING AND NON-LACTATING HOLSTEIN DONOR COWS

Embryo transfer is a common practice in reproductive management of large dairy herds. Data from 2 dairies in the San Joaquin Valley in California (5000 lactating Holstein producing 27 500 pounds of milk in 365 days) was used to evaluate the differences in embryo production between lactating (105.3 ± 24.6 pounds of milk/day, 161 ± 85 days in milk and 3 ± 1 lactations) and nonlactating donor cows, from July 2006 to June 2007. Cows were observed daily for estrous signs and superovulation treatment was initiated 10 d later. Cows received FSH twice a day during 4 days in decreasing doses. On the fifth treatment of FSH, cows received an injection of PGF2α and 48 h later an injection of GnRH. Cows were inseminated 12 and 24 h after the GnRH injection. Uterine flush was performed 7 d after the first AI only in cows that had 2 or more CL. The total number of ova/embryos, viable embryos (transferred fresh or frozen), degenerated, and unfertilized structures were recorded (classified according to the guidelines of IETS). A Bernoulli regression model was fitted using the R software (www.r-project.org) for the variable superovulation response and the covariates were cow condition (lactating or nonlactating), season, and interactions. From 719 superovulated cows, 668 were flushed (92.9%): 86.8% (309/356) of the lactating cows and 98.9% (359/363) of the nonlactating cows (P &lt; 0.01). There was evidence of season effect (P = 0.09). Cows tended to respond less to superovulation treatment during summer and fall. A negative binomial regression model was fitted to the response variables total number of ova/embryo and viable embryos and the covariates were cow condition, season, and interactions. Lactating cows produced fewer (6.77) ova/embryos than nonlactating cows (14.14; P &lt; 0.01). The adjusted means were 5.93 v. 12.53 for fall, 6.30 v. 13.32 for winter, 8.24 v. 17.40 for spring and 6.32 v. 13.34 for summer, for lactating and nonlactating cows, respectively. Cows produced more ova/embryos during spring (P &lt; 0.01). Less viable embryos were recovered from lactating cows (3.62) than nonlactating cows (5.44; P &lt; 0.01). The adjusted means were 2.52 v. 3.98 for fall, 3.61 v. 5.69 for winter, 4.51 v. 7.12 for spring and 3.37 v. 5.31 for summer, for lactating and non lactating cows, respectively. The production of viable embryos in fall was different from winter and spring (P &lt; 0.01). Lactating cows responded less to superovulation treatment and produced less viable embryos than nonlactating cows, probably because of the increased metabolism due to milk production. Warmer seasons were associated with lower viable embryo production. It is important to have a group of nonlactating donors to increase embryo production in large ET programs.