Age Of Embryo Research Articles

A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates.

OBJECTIVE:To evaluate the efficacy of blastocyst transfer in comparison with cleavage stage transfer.STUDY DESIGN:A randomized, prospective study was conducted in Infertility clinic, Department of Obstetrics and Gynecology, Mahatma Gandhi Hospital, Jaipur on 300 patients aged 25-40 years undergoing in-vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) cycle from May 2010-April 2011. When three or more Grade-I embryos were observed on day 2 of culture, patients were divided randomly into two study groups, cleavage stage transfer and blastocyst transfer group having 150 patients each. Primary outcomes evaluated were, Clinical pregnancy rate and Implantation rate. The results were analyzed using proportions, standard deviation and Chi-square test.RESULTS:Both the groups were similar for age, indication and number of embryos transferred. Clinical pregnancies after blastocyst transfer were significantly higher 66 (44.0%) compared to cleavage stage embryo transfer 44 (29.33%) (P < 0.01). Implantation rate for blastocyst transfer group was also significantly higher (P < 0.001).CONCLUSION:Blastocyst transfer having higher implantation rate and clinical pregnancy rate lead to reduction in multiple pregnancies.

Body mass index and pregnancy outcome after assisted reproduction treatment.

The present study aimed to evaluate the impact of body mass index (BMI) on pregnancy outcome after intracytoplasmic sperm injection (ICSI). The study analyzed pregnancy outcome of 349 women who underwent ICSI by their BMI: <25, 25–<30, and ≥30 kg/m2. The associations were generated by applying logistic regression models. A significant reduction in positive pregnancy outcome was observed among overweight and obese women (odds ratio (OR) = 0.50; 95% confidence interval (CI) = 0.25–0.99 for overweight women and OR = 0.45; 95% CI = 0.20–0.89 for obese women). These estimates show that the pregnancy rates are reduced with increasing BMI. The effect of obesity on pregnancy outcome was absent when three and more embryos were transferred. Our study contributes to the reports linking overweight and obesity with decreased positive pregnancy outcome after ICSI and suggests women's age, infertility type, and number of embryos transferred to modify this reducing effect.

INDUCTION OF DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE EMBRYOS OF BRASSICA OLERACEA VAR. SABAUDA L.

An efficient plant propagation system through somatic embryogenesis by using immature zygotic embryos was established in Savoy cabbage (Brassica oleracea var. sabauda L.). The effects of zygotic embryo age, presence of 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) (B5-D) in the culture medium, and the pH (5.0 and 5.8) of the B5 plant growth regulator (PGR)-free (B5-0) induction media were investigated. It appeared that the developmental stage of the immature zygotic embryos used as explants was the most important factor for somatic embryogenesis. Highest frequency of somatic embryogenesis (86.67%) was achieved directly on zygotic embryos at cotyledonary stage (1.8 mm long) of development that cultured on B5-0 medium. Optimal pH of the medium was 5.0. Mean number of primary somatic embryos per explant on B5-0 pH 5.0 medium was 7.2. The inclusion of 2,4-D into the induction medium significantly reduced both frequency of somatic embryogenesis (to 53.34 %) and mean number of embryos per explant (to 2.62) in cotyledonary zygotic embryos. After transferring to PGR-free Murashige and Skoog (MS-0) medium 69.69% of the primary somatic embryos of the Savoy cabbage produced secondary somatic embryos (SSEs) Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of Savoy cabbage.

Utilization of hysteroscopy in frozen-thawed embryo transfer (FET) cycles

FET is currently the main procedure for embryo transfer. Out of the total number of such procedures performed, those that did not result in pregnancy in the first trial were subjected to hysteroscopic examination. The usefulness of hysteroscopy was examined in the following comparisons, and in the subsequent procedure conditions that resulted in pregnancy. Retrospective study. A total of 241 pregnancies resulted out of 465 FET procedures (for a pregnancy rate of 51.8%) performed in 2012. Out of the total, cases that did not result in pregnancy in the first trial (n=142) were utilized as study subjects and divided into 3 groups. Group I: Patients that were not examined by hysteroscopy prior to a further transfer procedure being performed. Group II:Patients examined by hysteroscopy and diagnosed with various abnormalities such as inflammation, polyps, or poor vascularity and then treated by polypectomy or drug administration (L-arginine etc.) followed by a subsequent FET procedure. Group III:Patients that showed no uterine abnormalities upon examination by hysteroscopy and were directly treated with a subsequent FET procedure. The three groups had similar baseline characteristics; Endometrium thickness (>8mm for transfer), patient age, and good quality of embryos (Cleavage: GI, II, blastocyst >3BB) was evaluated in this study. Pregnancy rates in Group I, Group II and Group III were 40.4% (17/42), 60.3% (35/58) and 57.1% (24/42), respectively. Groups II and III had a significantly higher pregnancy rates compared to Group I (P<0.05). From the results of Group II, it was concluded that the pregnancy rate could be increased by treating patients with endometrial abnormalities that were identified by hysteroscopy. Furthermore, the pregnancy rate in patients examined by hysteroscopy with no identified abnormalities (GroupIII) also increased, which may be a result of the effects of saline solution perfusion during hysteroscopy.

Beyond the ASRM transfer guidelines: maximum number of embryos to transfer in women ≥ 43 years of age

To determine the maximum number of cleavage stage embryos that can be safely transferred in women ≥43 years old. Retrospective cohort. Cycles from 1/2004-4/2012 were reviewed for transfer of ≥5 day 3 embryos. Donor egg cycles were excluded. Implantation, clinical pregnancy, live birth, rate of twins, and rate of higher order multiples (HOM) were analyzed, stratifying by age and number of embryos transferred. Differences between groups were assessed via ANOVA with Bonferroni adjustment, where p<0.05 was considered significant. 561 patients ages 43-45 were identified whose IVF cycles were characterized by transfer of 5-8 cleavage stage embryos. No patient had more than 8 embryos transferred. 45 year old patients exhibited higher day 3 FSH than 43 year old patients. There were otherwise no demographic differences between ages. Each age group had experienced an average of 3 prior IVF failures. 2/66 (3%) of pregnancies in 43 year olds were triplet gestations, one of which selectively reduced to twins, and the other spontaneously reduced to twins. Twin birth was 11.7%, 6.8%, and 0% for ages 43, 44, and 45, respectively. There were no HOM births.Tabled 1Clinical outcomes for patients ≥43 undergoing transfer with ≥5 embryosAge 43# of Embryos5678n =151129126Implantation7.9%7.0%7.0%6.3%Live birth11.9%16.3%16.7%33.3%Twin FH23.5%23.3%0%50%Triplet FH2.9%3.3%0%0%% Twin Birth22.2%9.5%0%50%Age 44n =9278166Implantation4.4%5.6%0.9%4.2%Live birth10.9%7.7%6.3%16.7%Twin FH0%14.3%0%0%Triplet FH0%0%0%0%% Twin Birth0%16.7%0%0% Open table in a new tab Transferring as many as 8 cleavage-stage embryos can be viewed as a safe option for IVF patients ≥43 years of age with multiple prior failures.

The influence of houseflyMusca domesticaembryo age on viability in water and at low temperatures

The sensitivity of housefly Musca domestica L. (Diptera: Muscidae) embryos to storage at low temperatures (5 and 10 °C on moist sponges in Petri dishes) and in water at 26 °C was investigated to develop suitable protocols for the storage and transport of housefly eggs. The youngest embryos (aged 0-3 h) were the most sensitive to storage at 5 °C, with 45% survival after storage for 24 h. Storage of embryos aged 3-12 h at 5 °C for 24 h had no negative effect; longer storage resulted in significantly decreased larval survival (30-34% after 48-72 h, compared with 61% in the control group) and reduced hatching rates (83% after 72 h storage). No negative effects were observed when embryos aged 0-9 h were stored at 10 °C for 24 h, but this temperature did not completely inhibit development and eggs began to hatch if stored for longer than 24 h. All age groups of embryos showed high mortality after storage in water at 26 °C for 24 h, with the youngest embryos being least resistant to submersion.

Prenatal development of the agouti (Dasyprocta prymnolopha Wagler, 1831): External features and growth curves

The gestation period in agoutis can range from 104 to 120 days. Knowledge regarding the morphological characteristics of embryos and fetuses is important as a base for studies in reproduction biotechnology, such as in vitro fertilization, embryo transfer and helps in determining congenital anomalies during the development phase. Thus, given the importance and lack of information about agouti embryology, the objective of this study was to characterize the external morphology and define the biometry of embryos and fetuses, at different days of development. Nine females were submitted to daily colpocytology to identify the estrus, confirm mating and identify day zero of the gestation. When the mating was confirmed they were weighed, underwent abdominal ultrasonography and surgery was conducted on the females at the gestational ages of 25, 30, 35, 40, 45, 50, 75 and 100 days. Sixteen embryos/fetuses were weighed and measured. Agouti embryos at 25 days after mating are “C” shaped, with primitive structures, 0.4±0.01cm crown-rump and weighed 0.06±0.01g; at 30 days after mating the crown-rump was 0.95±0.07cm and weighed 0.28±0.00g; at 35 days after mating the crown-rump was 155±0.07cm and weighed 0.38±0.01g; at 40 days after mating the crown-rump was 2.25±0.21cm and weighed 1.25±0.07g; at 45 days after mating the crown-rump was 3.45±0.35cm and weighed 2.75±0.64g; at 50 days after mating the crown-rump was 5.0±0.3cm and weighed 7.01±2.6g; at 75 days after mating, the skin was dark, the crown-rump was 10.0±0.14cm and weighed 55.2±0.07g. At 100 days after mating, the crown-rump was 13.8±0.49cm and fetuses weighed 136.7±9.40g. Based on the morphological data assessed the embryo and fetus age could be assessed and the size and average weight of agouti embryos was established.

Abstracts from the Society for Acupuncture Research 2013 International ConferenceImpact of Acupuncture Research on 21st Century HealthcareApril 18–21, 2013The Michigan LeagueAnn Arbor, MI

Abstracts from the Society for Acupuncture Research 2013 International ConferenceImpact of Acupuncture Research on 21^st Century Healthcare<i>April 18–21, 2013</i><i>The Michigan League</i><i>Ann Arbor, MI</i>

Avian Incubation Patterns Reflect Temporal Changes in Developing Clutches

Incubation conditions for eggs influence offspring quality and reproductive success. One way in which parents regulate brooding conditions is by balancing the thermal requirements of embryos with time spent away from the nest for self-maintenance. Age related changes in embryo thermal tolerance would thus be expected to shape parental incubation behavior. We use data from unmanipulated Black-capped Chickadee (Poecile atricapillus) nests to examine the temporal dynamics of incubation, testing the prediction that increased heat flux from eggs as embryos age influences female incubation behavior and/or physiology to minimize temperature fluctuations. We found that the rate of heat loss from eggs increased with embryo age. Females responded to increased egg cooling rates by altering incubation rhythms (more frequent, shorter on- and off- bouts), but not brood patch temperature. Consequently, as embryos aged, females were able to increase mean egg temperature and decrease variation in temperature. Our findings highlight the need to view full incubation as more than a static rhythm; rather, it is a temporally dynamic and finely adjustable parental behavior. Furthermore, from a methodological perspective, intra- and inter-specific comparisons of incubation rhythms and average egg temperatures should control for the stage of incubation.

In Ovo Vaccination of Commercial Broilers with a Glycoprotein J Gene-Deleted Strain of Infectious Laryngotracheitis Virus

Conventional live attenuated vaccines have been used as the main tool worldwide for the control of infectious laryngotracheitis. However, their suboptimal attenuation combined with poor mass administration practices allowed chicken embryo origin vaccine-derived isolates to circulate in the field, regain virulence, and be the cause of continuous outbreaks of the disease. Previous studies indicated that stable attenuation of infectious laryngotracheitis virus (ILTV) can be achieved by the deletion of individual viral genes that are not essential for viral replication in vitro. One of these genes is the glycoprotein J (gJ) gene. Its deletion provided significant attenuation to virulent ILTV strains from Europe and the United States. The objective of this study was to construct an attenuated gJ-deleted ILTV strain and evaluate its safety and efficacy for in ovo (IO) administration of commercial broilers. A novel gJ-deleted virus (N(delta)gJ) was constructed, and a 10(3) median tissue culture infective dose administered at 18 days of embryo age was considered safe because it did not affect hatchability or survivability of chickens during the first week posthatch. Broilers vaccinated IO and IO + eye drop at 14 days of age presented a significant reduction in clinical signs and reduction of virus loads after challenge, as compared with the nonvaccinated challenged group of chickens. Therefore, this study presents initial proof that the N(delta)gJ strain is a potential ILTV live-attenuated vaccine candidate suitable for IO vaccination of commercial broilers.

A prospective randomized multicentre study comparing vaginal progesterone gel and vaginal micronized progesterone tablets for luteal support after in vitro fertilization/intracytoplasmic sperm injection

SUMMARY QUESTION: Is vaginal progesterone gel equivalent to vaginal micronized progesterone tablets concerning ongoing pregnancy rate and superior concerning patient convenience when used for luteal support after IVF/ICSI? Equivalence of treatments in terms of ongoing live intrauterine pregnancy rate has not been demonstrated; the 95% confidence interval (CI) for the difference in ongoing pregnancy rate (-8.2 to 0.1%) did not lie entirely within the pre-specified equivalence interval -7 to 7%. No significant differences in clinical pregnancy rates have been observed between vaginal progesterone gel and other vaginal progesterone products in earlier studies. However, all previous studies included a limited number of patients. This was a randomized, multicentre, controlled, assessor-blinded equivalence trial in 18 fertility centres in Denmark and Sweden between March 2006 and January 2010. A web-based randomization program was used with concealed allocation of patients. Patients were randomized to one of two groups: vaginal progesterone gel or vaginal micronized progesterone tablets. There was no blinding of patients. A total of 2057 women ≤ 40 years of age were included and down-regulated, using the long agonist protocol and rFSH for stimulation. Luteal support was given for 19 days after embryo transfer or until a negative pregnancy test Day 14 after embryo transfer. Patient convenience was assessed using questionnaires to be filled in 14 days after embryo transfer, before pregnancy test. Ongoing intrauterine pregnancy rates were 299/991 (30.2%) (95% CI 27.3-33.0%) in the progesterone gel group and 324/992 (32.7%) (29.7-35.6%) in the micronized progesterone tablet group. The difference in ongoing pregnancy rates between the groups was -4.1% (-8.2 to 0.1%) and the difference in live birth rates was -3.4% (-7.4 to 0.7%), both calculated after correction for significant confounders. Patient convenience and ease of use (1 = very convenient, 10 = very inconvenient) was in favour of progesterone gel, as the overall score was 2.9 (2.7-3.0) for progesterone gel and 4.8 (4.7-5.0; P < 0.0001) for micronized progesterone tablets. This large equivalence trial shows that, even though equality could not be demonstrated, there is no substantial difference in ongoing pregnancy rate between vaginal progesterone gel and vaginal micronized progesterone tablets. It also shows that progesterone gel is considered more convenient by the patients. Blinding of patients was not possible in this study, but since the outcome (pregnancy) is robust, blinding would have been unlikely to affect the results. Unfortunately, owing to an error in the randomization, the intended age distribution allocated older women to the micronized progesterone tablet group. In the analysis of results, adjustments were made for age and number of embryos transferred. The results can be generalized to other women ≥ 18 and ≤ 40 years of age undergoing IVF/ICSI who have regular menstrual cycles (25-35 days), both ovaries present and no more than two previous failed IVF attempts. Merck Serono supported the study but had no influence on the design of the study and was not involved in the analysis of the results or preparation of the manuscript. The trial was issued with the EudraCT number 2005-001248-22 with the Protocol code number 95576471.

Pregnancy loss after frozen-embryo transfer—a comparison of three protocols

To compare the reproductive outcome of three protocols for frozen ET treatment. Retrospective follow-up study. Two public clinics and one private clinic. Four thousand four hundred seventy frozen ET cycles between 2006 and 2010. Thawing of embryos and ET. Pregnancy test rate, clinical pregnancy rate, and pregnancy loss rate. The natural cycle followed by P (NC + P) was used in 26% of cycles, the natural cycle with hCG (NC + hCG) in 10%, and the substituted cycle with estrogen and P (E + P) in 64% of cycles. The rate of transfers after thawing was similar in all groups (87.2%, 73.9%, and 87.2%, respectively). There was a significantly higher positive pregnancy test rate in the E + P (34.3%) and NC + hCG (35.5%) cycles as compared with the NC + P cycles (26.7%). However, the clinical pregnancy rate was similar in all groups (27.7%, 29.1%, and 24.3%, respectively). Moreover, no differences were seen between groups regarding the live-birth rate (20.1%, 23.5%, and 20.7%, respectively). A logistic regression analysis showed that the type of protocol was the only predictor of pregnancy loss, while age, irregular cycles, endometrial thickness, number, and quality of embryos transferred did not correlate to pregnancy loss. A higher positive pregnancy test rate was obtained in E + P frozen ET cycles in comparison with other protocols; however, due to an increased preclinical and clinical pregnancy loss, comparable clinical pregnancy, and delivery rates are reported for the three protocols.

Developmental transition of pectoralis muscle from atrophy in late‐term duck embryos to hypertrophy in neonates

Unlike the mammalian fetus, whose growth is supported by the sustained provision of maternal nutrients, poultry embryos undergo development in a relatively closed space, and the yolk sac serves as the sole nutrient supply for embryonic development throughout the whole incubation period. To increase our understanding of the muscle developmental patterns in the final stage of incubation and early days posthatching, we used late-term duck embryos and newly hatched ducklings as animal models. Pectoralis muscle samples were collected at 22 days (22E) of incubation, 25 days (25E) of incubation, hatching and day 7 posthatching. The pectoralis muscle mass, muscle fibre bundles and myofibre cross-sectional area showed a marked reduction from 22E to hatching, but they increased dramatically by day 7 posthatching. The mRNA expression of Atrogin-1, a key mediator of the ubiquitin system responsible for protein degradation, increased dramatically with the age of late-term duck embryos, but it decreased by day 7 and reached a very low level. The extent of mRNA expression of FoxO1, one of the transcription factors of the Atrogin-1 gene, exhibited a transient increase at 25E and then decreased from hatching to day 7. The phosphorylated p70 ribosomal protein S6 kinase 1 (S6K1)/S6K1 ratio exhibited a dramatic reduction from 22E to hatching (P < 0.05) and then increased by day 7. The results of the present study indicated that there was a developmental transition of pectoralis muscle from atrophy in late-term duck embryos to hypertrophy in neonates.

Effect of temperature on embryonic development of Octopus mimus under controlled conditions

Although the mechanism by which temperature modulates the use of reserves and its consequences for organogenesis in octopus species is not yet known, there is evidence that temperature modulates the embryonic growth rate in cephalopods with consequences for hatchling characteristics. The primary objective of this study was to investigate the effect of temperature (12, 15, 18 and 21°C) on the duration of Octopus mimus embryo development and to generate a prediction model. The effects of temperature on size and physiological characteristics of embryos at stage XV were also studied. A third objective was to investigate whether temperature affects the time taken to reach stage XX using physiological time, i.e. degree-days (DD). The time taken to reach stage XV at 21°C was 24, 58 and 75% shorter than observed in eggs maintained at 18, 15 and 12°C, respectively. Logarithmic models were obtained for the relationship between embryo stage and age, demonstrating an inverse relationship between environmental temperature and the duration of embryonic development. Embryos maintained at 18 and 21°C grew faster than those kept at 12 and 15°C, although that growth rate was accomplished by an inverse relationship between yolk and temperature. The degree-day model suggests that between 128 and 133 DD should be accumulated when embryos are maintained at 15 to 21°C. As in other invertebrate species the results on oxygen consumption indicate that embryos of O. mimus have a higher sensitivity to temperature than adults. The results suggest that the optimum range of temperatures for these embryos is around 15 to 18°C.

Developmental transition of pectoral muscle from atrophy in late-term duck embryos to hypertrophy in neonates

Unlike the mammalian fetus, whose growth is supported by the sustained provision of maternal nutrients, poultry embryos undergo development in a relatively closed space, and the yolk sac serves as the sole nutrient supply for embryonic development throughout the whole incubation period. To increase our understanding of the muscle developmental patterns in the final stage of incubation and early days posthatching, we used late-term duck embryos and newly hatched ducklings as animal models. Pectoralis muscle samples were collected at 22 days (22E) of incubation, 25 days (25E) of incubation, hatching and day 7 posthatching. The pectoralis muscle mass, muscle fibre bundles and myofibre cross-sectional area showed a marked reduction from 22E to hatching, but they increased dramatically by day 7 posthatching. The mRNA expression of Atrogin-1, a key mediator of the ubiquitin system responsible for protein degradation, increased dramatically with the age of late-term duck embryos, but it decreased by day 7 and reached a very low level. The extent of mRNA expression of FoxO1, one of the transcription factors of the Atrogin-1 gene, exhibited a transient increase at 25E and then decreased from hatching to day 7. The phosphorylated p70 ribosomal protein S6 kinase 1 (S6K1)/S6K1 ratio exhibited a dramatic reduction from 22E to hatching (P < 0.05) and then increased by day 7. The results of the present study indicated that there was a developmental transition of pectoralis muscle from atrophy in late-term duck embryos to hypertrophy in neonates.

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.

95 SHEEP ZYGOTES CULTURED WITH HYALURONAN, BOVINE SERUM ALBUMIN, OR SERUM AND THEIR EFFECTS ON PRENATAL AND POSTNATAL EMBRYO PRODUCTION AND DEVELOPMENT

Mammalian pre-implantation embryos are sensitive to their environment and numerous publications suggest that post-fertilization culture is the most critical period affecting embryo quality and subsequent embryo development. This study was conducted to examine the viability, twining, sex ratio and birthweight (BW) after transfer of fresh and vitrified in vitro-produced sheep embryos derived from oocytes cultured in a semi- or defined medium supplemented with fatty-acid–free bovine serum albumin (BSA) with or without hyaluronan (HA) versus fetal bovine serum (FBS) as a control. A total of 649 oocytes were matured in TCM-199 containing 2 mM glutamine, 4 mg mL–1 BSA, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg mL–1 oestradiol-17β and fertilized with fresh semen prepared with SOF, enriched with 20% oestrus sheep serum. Presumptive zygotes were cultured for 48 h in SOF supplemented with 1% BME-essential and 1% MEM-nonessential amino acids, 1 mM glutamine and 8 mg mL–1 BSA. On the 3rd day of culture, presumptive zygotes were divided randomly into 3 groups: (1) BSA+HA, medium supplemented with 8 mg mL–1 BSA and 6 mg mL–1 HA, (2) BSA, medium supplemented with 8 mg mL–1 BSA only, or (3) control, medium supplemented with 5% charcoal-stripped fetal bovine serum (FBS). On the 5th day of culture, media were replaced accordingly in all groups. Embryos that reached the expanded blastocyst stage by 6 or 7 days/group were recorded and transferred as fresh or vitrified. The cleavage rate, blastocyst formation, pregnancies (PR) at 40, 60 and 80 days, lambing, twinning, sex ratio and BW were all recorded. Among all groups, no significant differences were observed in cleavage and blastocyst rates. The proportion of zygotes that developed to blastocysts at 6 days was significantly higher after culture with BSA+HA vs BSA and control (67/92, 72.8%), (46/118, 39.0%) and (29/72, 40.3%), respectively. Compared with the control group, vitrified BSA blastocysts were associated with increased (P &lt; 0.05) PRs after 60 and 80 days and lambing rates. In comparison, there were no significant differences in pregnancy and lambing rate after transfer of fresh blastocysts. There was a positive correlation between age of embryo at transfer and higher BW. The mean BWs were higher after transfer of 7-day vs 6-day embryos (3.95 and 3.37 kg), respectively. However, there were no significant differences in twining and sex ratio after transfer fresh/or vitrified treated embryos versus control group. In conclusion, the results indicate that sheep zygotes cultured with BSA+HA had an increased development to blastocysts at 6 days, but the rate of blastocyst formation was not increased, PR and lambing rate were comparable with the BSA and control groups. In addition, transfer of vitrified embryos produced in the presence of BSA significantly increased the PR at 60, 80 days and lambing rate versus control group.

108 THE EFFECTS OF FORSKOLIN ON BRAHMAN IN VIVO-PRODUCED EMBRYOS AND SUBSEQUENT PREGNANCY RATES POST-THAW

It has been hypothesised that lower survival rates post-thaw of Brahman (BR) embryos is due in part to higher intracellular lipids. The objective of this study was to determine whether pregnancy rates of in vivo–produced BR embryos could be enhanced by exposing them to 10 μM of forskolin (FSK; lipolytic agent) before slow cool freezing, thawing and direct transfer. A previous report (Phase II) indicated that the addition of FSK to culture 24 h before freezing 7 days in vitro–produced, BR-sired embryos can increase survival and blastocyst hatching rates (Pryor et al. 2010 Reprod. Fertil. Dev. 22, 214). For this study (Phase III), 11 BR cows were injected with 25 mg of Lutalyse® (Pfizer Animal Health, New York, NY, USA) to synchronize oestrus. On Day 0 (7 days after synchronized oestrus), all donors were inserted with a CIDR (Controlled Intravaginal Releasing Device; Pharmacia Co., Kalamazoo, MI, USA) and injected with 50 mg of progesterone plus 2.5 mg of oestradiol 17 β (Medshop, Longview, TX, USA). On Day 4, decreasing doses of Folltropin (Bioniche, Pullman, WA, USA) ranging from 160 to 264 mg were given twice daily for 3 days along with 0.625 mg of cloprostenol (Estrumate; Merck/Schering-Plough Animal Health, Whitehouse Station, NJ, USA) given twice on Day 6 (AM and PM) and CIDR removal on Day 7 AM. Donors were artificially inseminated 12 and 24 h post-oestrus (oestrus = Day 8) with frozen/thawed BR semen. On Day 14 (embryo age 6 days), donors were nonsurgically collected, producing a total of 75 grade-one morulae, which were randomly allocated and cultured for 24 h in Evolve (Zenith Biotechnology, Canada) supplemented with 4 mg mL–1 of Probumin BSA (Millipore, Norcross, GA, USA; control) or with FSK (Sigma, St. Louis, MO, USA) at 38.5°C under a 5% CO2, 5% O2 and 90% N2 humidified atmosphere. Immediately following treatment, 7 days compact morula/blastocysts were washed in Vigro Holding Plus medium (Bioniche, Belleville, Ontario, Canada) and submitted to Vigro Ethylene Glycol Freeze Plus medium (Bioniche) for 5 to 7 min before being frozen at 0.5°C min–1 from –6°C to –32°C and plunged in liquid nitrogen. Frozen embryos (n = 35 FSK; n = 35 control) were air thawed for 7 s and then immersed in 30°C H2O for 10 s before being nonsurgically transferred into synchronized recipients. Pregnancy rates were assessed by ultrasonography via rectal palpation 30 to 60 days post-transfer. Contingency analysis was performed using forskolin treatment, technician and embryo stage and location as independent variables (JMP 8.0, SAS Institute Inc., Cary, NC, USA). There were no statistical differences or interactions for any of the analysed variables. Pregnancy rates between control and FSK treatments did not vary (34.3 and 31.4%, respectively; chi-square P = 0.80). In conclusion, treating in vivo–produced BR embryos with 10 μM of forskolin for 24 h did not alter pregnancy rates. The authors acknowledge support from the American Brahman Breeders Association.

105 BOVINE EMBRYO TYPE, GRADE AND RECIPIENT SYNCHRONY, BUT NOT CORPUS LUTEUM QUALITY, INFLUENCED PREGNANCY RATES IN COMMERCIAL EMBRYO TRANSFER

Embryo transfer is a tool that enables progressive cattle producers to reach their reproductive, genetic and financial goals. The complexity of pregnancy establishment increases with any reproductive technology, especially with one with many variables. This clinical analysis employed a statistical model capable of explaining factors that affect pregnancy probability resulting from over 8000 embryo transfers. These data included corpus luteum (CL) quality score, embryo stage (morula to blastocyst) and grade (1–3), recipient synchrony (relative to the embryo age) and pregnancy rate (≥45 days). The CL score was an amalgamation of the palpable diameter of the CL, whether or not a crown was present and the overall quality of the reproductive tract (size of the uterine horns and perceived patency of the cervix). Embryos produced by IVF were transferred fresh and in vivo–produced embryos were transferred fresh (VIVO FRESH) or following cryopreservation (VIVO FROZ). Statistical methods were used to compare types of embryos transferred and pregnancy rates. All data were analysed using JMP 2009 software by logistic regression with the Logit link. Analysis for the odds of pregnancy occurring among groups using contrasts was conducted. Fisher's exact test showed significant differences (P &lt; 0.05) among groups for pregnancy rate, with the VIVO FRESH embryos group having the highest rate (68.5 ± 1.5%) and the VIVO FROZ the lowest (54.7 ± 1.6%) and the IVF embryos were intermediate (62.1 ± 3.3%). Synchrony, stage, embryo grade and CL quality were analysed for their predictive ability of pregnancy rates in the IVF, VIVO FRESH and VIVO FROZ groups using effect tests with a P &lt; 0.05. Interestingly, the CL quality had no predictive ability for any of the groups. Effectors that had an influence on the VIVO FROZ group included synchrony, stage and embryo grade. However, for the IVF and VIVO FRESH groups, pregnancy rates were affected by only embryo grade. Retrospective, observational analysis of commercial embryo transfer data represents a challenge to meaningful interpretation. Because of the nature of the business, experimental treatments cannot always be developed and implemented. Nonetheless, valuable information is gained by using appropriate statistical methods. As anticipated, these data suggested that the type of embryo transferred, grade and synchrony of the recipient are all important factors, particularly for cryopreserved embryos. Unexpectedly, the score of the CL did not influence pregnancy outcome regardless of embryo type. Evaluation of clinical data allows adjustment of embryo transfer protocols, facilitates genetic gain via enhanced reproductive success and provides a unique opportunity to analyse large, real-world data sets.

Clinical and economic analysis of rescue intracytoplasmic sperm injection cycles

Objective. To identify clinical and embryological factors that may predict success in rescue intracytoplasmic sperm injection (ICSI) cycles (after total fertilization failure has occurred) and to evaluate the cost effectiveness of rescue ICSI strategy. Additionally, follow-up of 20 rescue ICSI pregnancies is reported.Design. Retrospective analysis of total fertilization failure cycles.Setting. University-based tertiary medical center.Material and methods. In total, 92 patients who had undergone conventional in-vitro fertilization (IVF) cycles with total fertilization failure were included. The patients were divided into two subgroups: those who conceived through rescue ICSI and those who did not.Results. The pregnant members of the rescue ICSI subgroup were found to be significantly younger (32.9 ± 4.2 vs. 36.3 ± 4.5, respectively, p = 0.0035,) and to have better-quality embryos than those who did not conceive (cumulative embryo score: 38.3 ± 20.4 vs. 29.3 ± 14.7, p = 0.025). Cost effectiveness analysis showed 25% reduction in the cost per live birth when rescue ICSI is compared to cycle cancellation approach. The pregnancies follow-up did not show adverse perinatal outcome.Conclusions. Rescue ICSI is an option for salvaging IVF cycles complicated by total fertilization failure. Success in rescue ICSI was found to be associated with younger age and higher quality of embryos. Furthermore, the cost effectiveness of rescue ICSI in terms of total fertilization failure was found to be worthwhile.