Age Of Embryo Research Articles

Broiler Breeder Trace Mineral Nutrition and Feeding Practices on Embryo Progeny Development

This study was conducted to evaluate the effects of breeder feeding programme, feeder space change from rearing to lay, and trace mineral (TM) source on the development of embryo progeny. Cobb 500 broiler breeders were raised in a blackout pullet house. At 23 weeks, females that represented the body weight (BW) distribution from each pen were transferred to a laying house divided into pens with either same or more feeder space than in rearing. From 14 to 29 weeks, either a late fast or a late slow FP was used. Breeders were fed corn based diets during rearing and lay and from 56 to 62 weeks of age, these diets contained either 100% inorganic TM or an organic TM source to replace 30% of Cu, Zn, and Mn. Embryo BW, residual yolk, and gastrointestinal tract (GIT) development from 19 to 21 days of incubation were evaluated in 62-weeks-old breeder offspring. Allometric coefficients were determined for each GIT component. All possible interactions of this 2 × 2 × 2 factorial design were evaluated at each embryo age. Results indicated treatment effects on relative weights of GIT organs for only two-way and main effects. Allometric coefficients suggested that progeny of breeders fed diets with 100% inorganic TM or according to late fast (LF) had faster development of gizzard + proventriculus and gut, respectively. In breeder feeding practices and TM nutrition may affect GIT development of embryo progeny.

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish

Efficiency of preimplantation genetic screening (PGS) using array-CGH compared to matched control IVF patient populations with and without day-3 PGS fish

An investigation of the chick chorioallantoic membrane as an alternative model to various biological tissues for permeation studies

The chick chorioallantoic membrane (CAM) was explored as a biological membrane for use in the study of drug permeation with a Franz diffusion cell. The CAM was removed from fertilized chicken eggs of embryo age 9-18 days. The permeation profiles of nicotine through the fresh CAM were first obtained with a Franz diffusion cell. The permeation profiles of nicotine through frozen CAM, snake skin, pig skin, pig retina and pig buccal mucosa were also determined and compared with those of the fresh CAM. The permeability coefficient of the CAM varied with its age. The CAM at embryo age 13 was the most robust, showing the lowest standard error in permeability. It was thus chosen for comparative studies with snake skin, pig skin, retina and buccal mucosa. The CAM was found to be most similar to the buccal mucosa in terms of permeation profile and permeability coefficient values. Frozen CAM was also found to have a higher permeability coefficient than fresh CAM. The enhanced permeability was attributed to freezing, which affected the integrity of the CAM structure. From the findings, CAM shows potential as an alternative to the pig buccal mucosa as an in-vitro buccal model. The robustness of the CAM for drug permeation studies is affected by its age.

Symmetry in number of retrieved oocytes between two ovaries: a possible predictor of in vitro fertilization outcome

Objective. To examine the relationship between the ratio of difference in number of retrieved oocytes from the two ovaries to total number of oocytes (difference ratio, |NRO−NLO|/NTOTAL) and in vitro fertilization (IVF) outcome.Methods. Retrospective review of medical record for infertility patients with intact two ovaries who had undertaken controlled ovarian stimulation (COS) and IVF cycles in Seoul National University Hospital was conducted. Characteristics and IVF outcomes were compared according to the difference ratio (|NRO−NLO|/NTOTAL) using student t-test and χ2 test. To adjust for the effect of confounding factors, binary logistic regression test was performed.Results. When the study population was divided according to the difference ratio by cut-off value of 0.4, patients with difference ratio less than 0.4 showed significantly higher pregnancy rate (25.7% vs. 17.1%, p = 0.021). A significant correlation was noted between the difference ratio and achievement of pregnancy after adjustment for patient's age and total number of top quality embryos transferred (adjusted OR = 0.40, 95% CI 0.19–0.83, p = 0.014).Conclusions. The difference ratio of retrieved oocytes in patients with intact two ovaries was observed to have prognostic value in IVF cycles.

Laser-assisted Vitrification of Large Equine Embryos

The major difficulty in providing the benefits of embryo cryopreservation for equine agriculture is the mismatch between the optimal embryo age for collection from the mare (7-8 days after ovulation was detected) and the optimal age for freezing under current methods (6.5 days after ovulation). To overcome this limitation, we tested a method to enhance penetration of cryopreservative across the capsule and trophoblast of day 7 and 8 embryos combined with rapid freezing by vitrification. Six small embryos (<300 μm in diameter) were collected on day 6-7 after ovulation and twelve larger embryos were recovered on day 7-8. In the treatment group, replacement of blastocoelic fluid with cryopreservative solution was facilitated by a laser system used to create a small opening in the embryonic capsule and trophectoderm. All embryos were vitrified using a CryoLeaf freezing support. After recovery from freezing and embryo transfer, three of four small untreated embryos (<300 μm in diameter, 75%) and four of nine large blastocysts in the treatment group (>300 μm in diameter, 44%) resulted in a vesicle as detected by ultrasonography approximately one week after transfer. However, only one recipient mare was still pregnant on day 23, and she delivered a live foal. Further investigation is required to determine why most of the embryos in this experiment were lost between day 13 and day 23 of gestation.

Morphogenesis of the human palma arch using three-dimensional geometric modeling

The hand goes through complex morphological modifications during embryogenesis. The goal of this study was to use geometric modeling to study the morphometric modifications of the palmar arch. Five embryos were used for the study (sizes: 15, 17, 23, 30, and 44 mm). After digitalization of histologic sections (Sony DXC-930P 3CCD camera, Leica Qwin) and segmentation of the metacarpal cartilaginous matrices (Winsurf 4.3 software), geometric modeling and calculations were performed using MSC.Patran 2005r2 software. Correlations (r > 0.99) were found between embryo size and metacarpal volume, metacarpal surface, and the surface of the modeled palmar arch. The growth of the palmar arch is nonhomothetic. Significant reduction (P = 0.05) in the divergence of the 2nd, 3rd, 4th, and 5th metacarpals was observed. Deepening of the palmar arch is correlated with embryo size and age (r > 0.99). Geometric modeling allows 3D rendering of histologic sections and thus quantitative description of the morphogenesis. The results of this study support the hypothesis that opposition of the thumb in correlation with deepening of the palmar arch appears early in embryological development. It constitutes a specific morphological characteristic that appears very early in the human phylum. The fact that the human thumb is naturally in opposition in the resting position is a consequence of this morphogenesis. The thumb's resting position has received little attention in clinical settings and should be considered as the reference position for biomechanical analysis of the thumb column.

Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines (W21019, B7, and QCL5036) were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L −1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect ( P<0.05) on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L −1 (B7 and QCL5036) to 3.0 mg L −1 (W21019). The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L −1 of 6-benzylaminopurine (6-BA). 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6–0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

Aneuploidy Patterns in 3143 day-3 Embryos Analyzed by Array CGH

BACKGROUND: Previous FISH studies with day-3 biopsy showed an excess of monosomies, a correlation between maternal age and percentage of normal embryos (lower maternal age = higher percentage of normal embryos), and no correlation between maternal age and mosaicism. These studies were performed with 9-12 chromosome probes and thus did not include more than half of the chromosome complement. OBJECTIVE(S): To assess the types of chromosome abnormalities observed by array CGH and their relationship to maternal age. MATERIAL AND METHOD(S): Single blastomeres from day-3 embryos were analyzed by aCGH (24Sure, BlueGnome, UK). This technique can analyze all chromosomes with a very low error rate, including errors caused by mosaicism, (2%, Gutierrez-Mateo et al. 2010) and thus we assume that day-3 PGD results are mostly representative of the rest of the embryo. An embryo with 3 or more chromosomes out of the 24 being abnormal was considered “complex abnormal”. RESULT(S): Complex abnormal embryos and double aneuploidies significantly increased with advancing maternal age, while trisomies and monosomies did not. Normal embryos significantly decreased with advancing maternal age (48% in the <35 years of age group to 21% in the >39 years). We observed an excess of monosomy events (n=1006) compared to trisomy events (n=695) with one third of that excess being caused by monosomy X. There was a slightly higher average maternal age for aneuploidy events on chromosomes 13 to 22 (av. Age 38.4) than 1 to 12 (37.7), while monosomy X results showed an average maternal age of 36.4 years. Normal embryo results had an average maternal age of 35.9 years, compared to complex abnormal embryo results of 38.7 years. The most common aneuploidies were for chromosomes 16 (n=204), 22 (157), X (125), 15 (119), 21 (107), and 19 (91). CONCLUSION(S): Large studies using FISH (Munne et al. 2007) considered complex abnormalities akin to chaotic mosaics. However, by analyzing all chromosomes with aCGH, many embryos classified by aCGH as complex abnormal must be true aneuploid, as seen by their increased maternal age. The percentage of abnormal embryos does increase significantly with maternal age, as does the percentage of double aneuploids and complex abnormals. The excess of monosomies observed is not an artifact, as described in a separate abstract by Colls et al. SUPPORT: Departmental funds

Effect of Patient Age and Embryo Parameters on Pregnancy Outcome in In Vitro Fertilization-Embryo Transfer (IVF-ET)

Objective To study the effect of patient age, the number and quality of embryos transferred on pregnancy outcome in in vitro fertilization-embryo transfer procedures (IVF-ETs). Methods A retrospective study was conducted with infertile women who underwent a total of 1 800 cycles of IVF-ET and intracytoplasmic sperm injection (ICSI) at the Reproductive Medicine Center of the Third Affiliated Hospital of Guangzhou Medical College from Jan. 2006 to Dec. 2007. The patients were divided into three groups based on age (year): Results 1) In the group of patients aged Conclusion In an effort to achieve the ideal pregnancy rate without the risk of multiple pregnancies, it is desirable to employ a single good-quality embryo transfer for patients aged

Preimplantation genetic screening on patients with meiotic disorders

OBJECTIVE: Meiotic disorders increase the percentage of sperm carrying chromosome abnormalities that can rise the rate of abnormal embryos. The major difficulty in diagnosing meiotic disorders is that they can only be directly diagnosed trough the study of meiosis in testicular biopsies. The two aims of this study have been: 1)To compare the aneuploidy rate in embryos of males with meiotic disturbances with a control group; 2) To analyze the outcomes obtained after the Preimplantation Genetic Screening (PGS) cycles. DESIGN: Prospective cohort study. MATERIALS AND METHODS: The study included 32 patients whose evaluation of meiotic stages, chiasmata count and meiotic characterization resulted abnormal. The analysis was performed in classic meiotic preparations of processed unilateral testicular biopsy. A single blastomere from 185 embryos coming from 42 treatment cycles was evaluated, being abnormal meiosis the only indication for PGS. Biopsy was performed on day 3 embryos and FISH was assessed for chromosomes 13,15,16,18,21,22, X and Y. A control group of 31 fertile couples performing 33 cycles with 200 embryos evaluated was used for statistical comparisons. This group underwent PGS for sex-linked diseases. Fisher's exact tests with Yates correction were used for statistical analysis. RESULTS: When male had abnormal meiosis there was a higher percentage of aneuploid embryos than the control group (57.46 vs 34.0%, p<0.01) gonosome abnormalities (15.47% vs 7.53% p=0.02) and trisomies 13 (4.32% vs 0.05%, p=0.03). However trisomies 21, trisomies 18, haploidy, triploidy and tetraploidy rates were similar. Regarding PGS cycles, both groups obtained comparable clinical pregrancy rate, implantation rate and miscarriage rate (47.06% vs 40.00%; 42.62% vs 34.40%; 12.5% vs 16.7%) being women's mean age and number of embryos replaced similar. CONCLUSION: Meiotic abnormalities have a direct effect on the chromosomal constitution of embryos. PGS should be offered to these patients, because it provides high pregnancy and implantation rate.

Assessment of male serum anti-Mullerian hormone as a marker of spermatogenesis and ICSI outcome

Objective. To assess serum anti-Mullerian hormone (AMH) as a marker of spermatogenesis among fertile and infertile males, as well as its relation to ICSI outcome.Methods. A total of 77 male partners of infertile couples seeking infertility treatment were recruited for this study. They were classified according to the WHO criteria of semen analysis into three groups; azospermia, oligospermia, and normal. All participating patients had a serum assay of the level of AMH. Thirty-three couples out of the previously mentioned 77 couples underwent controlled ovarian stimulation and ICSI.Results. There were 41 patients with azospermia, 14 patients with oligospermia, and 22 patients with normal count. There was no significant difference among the three groups regarding the AMH levels. There was no significant correlation between the AMH levels from all patients and the sperm concentration (rho = 0.03, p = 0.82). Among patients who underwent ICSI, there was no significant correlation of the AMH with the age, sperm concentration, fertilisation percent or number of embryos. The age of male partners was significantly correlated with sperm concentration, fertilisation percent and the total number of embryos. In the logistic regression model used, serum AMH had no significant relation to clinical pregnancy.Conclusion. Male serum AMH levels are not indicative of spermatogenesis and cannot differentiate between fertile and infertile males. Serum AMH levels were not predictive of ICSI outcome as well.

Transfer of a selected single blastocyst optimizes the chance of a healthy term baby: a retrospective population based study in Australia 2004-2007

The practice of single embryo transfer (SET) is highly accepted by clinicians in Australia. This study investigates whether the SET of blastocysts results in optimal perinatal outcomes. This retrospective population-based study included 34 035 single or double embryo transfer cycles in women who had their first fresh autologous treatment in Australia during 2004-2007. Pregnancy, live delivery and 'healthy baby' (live born term singleton of > or = 2500 g birthweight and survived for at least 28 days without a notified/reported congenital anomaly) rates per transfer cycle were compared in four groups: selective single embryo transfer (SSET), unselective single embryo transfer (USSET), selective double embryo transfer (SDET) and unselective double embryo transfer (USDET). Live delivery and 'healthy baby' rates per transfer following SSET were further compared by number of embryos available. The analysis was stratified by woman's age and stage of embryo development. The highest rates of live delivery and 'healthy baby' per transfer cycle (46.2 and 38.0%) were achieved with transfer of a single blastocyst in women aged younger than 35 years. In women aged younger than 40 years, SSET had a significantly higher rate of 'healthy baby' per transfer cycle than did SDET regardless of stage of embryo development. In woman aged younger than 35 years who had SSET, there was no significant difference in live delivery and 'healthy baby' rates per transfer cycle whether two, three, four or five embryos were available. For all of these women, SSET of a cleavage embryo had significantly lower rates of live delivery and 'healthy baby' per transfer cycle compared with SSET of a blastocyst where only two blastocysts were available. Consultation with the patient with respect to the advantage of extended culture and selective single blastocyst transfer will result in better success rates following assisted reproductive technology treatment in Australia.

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.

Somatic embryogenesis and plant regeneration of tropical maize genotypes

In Latin America and sub-Saharan Africa, tropical maize (Zea mays L.) is a major crop for human consumption. To cope with the increasing population and changing environment, there is a need for improving tropical maize germplasm. As part of a biotechnological approach, efficient in vitro regeneration of two tropical maize inbred lines (CML216 and CML244) was established. A number of parameters were optimized, such as age of the immature embryos, plant media and growth regulator concentration. After 6 weeks of culture, somatic embryos that had already reached the coleoptilar stage produced shoots after light induction and developed into fertile plants after acclimation in the soil. The callus induction frequencies and somatic embryo-derived plantlet formation were higher when cultured with the Linsmaier and Skoog medium than those with the Chu’s N6 basal medium. Regeneration of tropical maize shoots depended on the 2,4-dichlorophenoxyacetic acid (2,4-D) concentration at the callus initiation stage from immature embryos. The recalcitrance of the tropical maize inbred line TL26 to in vitro regeneration was overcome in a single-cross hybrid with the CML216 and CML244 genotypes. Remarkably, tropical maize somatic embryos were formed at the abaxial side of the scutellum facing the medium, probably from the axis of the immature embryos, as shown by histological sections. Upon co-cultivation, agrobacteria transiently expressed their intronless β-glucuronidase-encoding gene at the embryogenic tissue, but not with an intron-containing gene, suggesting that virulence genes are induced in Agrobacterium, but that subsequent steps in the T-DNA transfer are inhibited.

Toxicity of dispersed weathered crude oil to early life stages of Atlantic herring (Clupea harengus)

Reports of the chronic toxicity of dispersed crude oil to early life stages of fish perpetuate uncertainty about dispersant use. However, realistic exposures to dispersed oil in the water column are thought to be much briefer than exposures associated with chronic toxicity testing. To address this issue, the toxicity of dispersed weathered oil to early life stages of Atlantic herring (Clupea harengus) was tested for short exposure durations, ranging from 1 to 144 h. Toxicity was a function of concentration and duration of exposure, as well as of the life stage exposed. Medium South American crude oil dispersed with Corexit 9500 caused blue sac disease in embryos, but not in free-swimming embryos. The age of embryos was negatively correlated with their sensitivity to oil; those freshly fertilized were most sensitive. Sensitivity increased after hatch, with free-swimming embryos showing signs of narcosis. Gametes were also tested; dispersed oil dramatically impaired fertilization success. For exposures of less than 24 h, gametes and free-swimming embryos were the most sensitive life stages. For those of more than 24 h, young embryos (<1 d old) were most sensitive. The results are presented as statistical models that could assist decisions about dispersant use in the vicinity of fish spawning habitats.

Efficient plant regeneration from immature embryo cultures of Jatropha curcas, a biodiesel plant

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1–1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog’s (MS) medium supplemented with IBA (0.5 mg l−1) and BA (1.0 mg l−1). The above medium when supplemented with growth adjuvants such as 100 mg l−1 casein hydrolysate + 200 mg l−1l-glutamine + 8.0 mg l−1 CuSO4 resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 mg l−1 polyvinyl pyrrolidone + 30 mg l−1 citric acid + 1 mg l−1 BA + 0.5 mg l−1 Kn + 0.25 mg l−1 IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-strength MS medium supplemented with 0.5 mg l−1 IBA and 342 mg l−1 trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.

Long-term survival and development of fetal ventral spinal grafts into the motoneuron-depleted rat spinal cord: Role of donor age

Fetal spinal cord (SC) tissue grafts can survive and develop into the lesioned SC, but no conclusive data are available concerning the long-term fate of transplanted material and the relation between the graft fate and the donor embryo age. Here, pre-labelled suspensions of ventral SC from E12 or E17 rat fetuses were grafted to the lumbar SC of adult rats with motoneuron depletion induced by perinatal injection of volkensin. E12 and E17 are presumably the stages when motoneuron development starts and terminates, respectively. Four or 10 months post-grafting, SCs were analyzed to check the graft survival rate and to follow the differentiation and spatial distributions of grafted cells. Neurotoxic lesion produced a 61% motoneuronal loss in the lumbar SC. In transplanted animals, all E12 fetal grafts survived until the observed time-points and developed various mature cell phenotypes. Many motoneuron-like labelled cells were found within the graft area or adjacent to it. Conversely, none of the E17 fetal grafts survived, since no graft-derived elements with neuronal morphology were found either in the site of graft placement or adjacent to it. The present findings indicate that spinal neuroblasts can survive for a long time and develop within the motoneuron-depleted SC, and that the donor embryo age is crucial for successful engraftment.

Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys

Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds.

Accuracy of Egg Flotation Throughout Incubation to Determine Embryo Age and Incubation Day in Waterbird Nests

Floating bird eggs to estimate their age is a widely used technique, but few studies have examined its accuracy throughout incubation. We assessed egg flotation for estimating hatch date, day of incubation, and the embryo's developmental age in eggs of the American Avocet (Recurvirostra americana), Black-necked Stilt (Himantopus mexicanus), and Forster's Tern (Sterna forsteri). Predicted hatch dates based on egg flotation during our first visit to a nest were highly correlated with actual hatch dates (r = 0.99) and accurate within 2.3 ± 1.7 (SD) days. Age estimates based on flotation were correlated with both day of incubation (r = 0.96) and the embryo's developmental age (r = 0.86) and accurate within 1.3 ± 1.6 days and 1.9 ± 1.6 days, respectively. However, the technique's accuracy varied substantially throughout incubation. Flotation overestimated the embryo's developmental age between 3 and 9 days, underestimated age between 12 and 21 days, and was most accurate between 0 and 3 days and 9 and 12 days. Age estimates based on egg flotation were generally accurate within 3 days until day 15 but later in incubation were biased progressively lower. Egg flotation was inaccurate and overestimated embryo age in abandoned nests (mean error: 7.5 ± 6.0 days). The embryo's developmental age and day of incubation were highly correlated (r = 0.94), differed by 2.1 ± 1.6 days, and resulted in similar assessments of the egg-flotation technique. Floating every egg in the clutch and refloating eggs at subsequent visits to a nest can refine age estimates.

136 EARLY EMBRYONIC LOSSES USING PROGESTERONE SUPPLEMENTATION IN CYCLIC AND NONCYCLIC RECIPIENT MARES

The aim of the present study was to determine the effectiveness of the commercial progesterone to maintain the pregnancies in noncyclic recipient mares and if it helps to decrease the early embryonic death when used in cyclic pregnant recipient mares. A total of 199 mares were used during February to April of the 2008 breeding season at Burns Ranch. The mares were assigned to groups according to their reproductive status. Noncyclic (n = 42) mares were prepared as embryo transfer recipients using our standard protocol with estradiol cypionate (3 days, 10/6/4 mg, Wickliffe, USA) and long action progesterone (P4LA, 1500 mg, BET Labs, Lexington, KY, USA), and 157 naturally cyclic mares were used from Day 3 to 8 after ovulation as embryo transfer recipients. After embryo transfer, 134 of the 157 cyclic mares got pregnant at 15 days. Some of the pregnant mares (n = 43) were assigned to a progesterone-supplemented group; these mares were administered P4LA (1500 mg) after the first pregnancy diagnosis at around 15 days and repeated 7 days later. The remaining mares were placed in a control group (n = 91), with no treatment, in order to compare the embryonic loss rates for the 3 groups. Recipients were evaluated for the presence of the embryonic vesicle between 13 and 16 days (embryo age), and pregnancies were monitored at 30 and 45 days to determine the early embryonic loss. Statistical analyzes were performed using the chi-square test with 5% defined as significance. Pregnancy rates at 13 to 16 days were not different (P = 0.336) for noncyclic and cyclic recipients (39/42, 90.7% and 134/157, 85.4%, respectively). The embryonic loss was evaluated in the 3 groups as follows: control group; 7 of 91 pregnancies were lost between 15 and 30 days (7.69%) and 2 of 84 were lost between 30 and 45 days (2.38%); the progesterone-supplemented group had lost 4 of the 43 pregnancies between 15 and 30 days (9.30%); and the noncycling progesterone mares had lost 2 of the 39 between 30 and 45 days (5.13%). Rates of embryo loss before 45 days of gestation were not different for the control mares with no progesterone supplementation (9.89%), progesterone-supplemented cyclic mares (9.3%), and noncyclic recipients (5.13%). The noncyclic recipients maintain gestation with the supplemental progesterone with no influence on embryo loss. These results demonstrate no advantage in the use of exogenous progesterone to supplementation for cyclic embryo recipients. Burns Ranch Inc., Menifee, California, USA.