Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos

140 INTERFERON-τ SECRETION BY IN VIVO DERIVED BOVINE TROPHOBLASTIC VESICLES

In ruminants, interferon-τ (IFN-τ) is a major pregnancy factor, secreted by the embryonic trophoblast cells during the pre-implantation period, being important for the maternal-fetal recognition. The co-transfer of bovine trophoblastic vesicles (bTVs) derived from in vivo recovered conceptuses is known to promote the successful implantation of embryos with expected lower viability, such as in vitro handled embryos, through the effects of IFN-τ secreted by bTVs. We have also reported that the pregnancy rate was improved using this technique in early pregnancy phase (Hashiyada et al. 2005 J. Reprod. Dev. 51, 749-756). However, the IFN-τ secretion level from bTVs has not been well known. Therefore, the objective of the present study was to measure concentration of IFN-τ released from individually cultured bTVs in vitro. Furthermore, we also investigated the transition of IFN-τ level in continuous culture of bTVs. Blastocysts were produced by artificial insemination of Japanese black cows following superstimulatory treatment and were recovered on 16 or 18 days post-estrus. Sixty-eight bTVs were prepared from 23 elongating blastocysts, 3 to 20 mm in length, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of 96-well plates using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 24 h of culture, fragments of unformed vesicles were re-cultured for an additional 24 h; 10 bTVs from this group were continuously cultured until Day 24 (the day of insemination was defined as Day 0). The volume of culture medium was 100 μL/well/day until Day 2 and thereafter changed to 200 μL/well/2 days to terminate. The viability of bTVs was assessed based on maintained spherical shape of vesicle, morphologically. Exchange and collection of culture media, morphological observation of bTVs were performed on Days 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Culture fluids were stored at -30°C. IFN-τ was measured by RIA (Takahashi H et al. 2005 Theriogenology 63, 1050-1060). Data were analyzed by Student’s τ-test Initial IFN-τ secretion did not differ between groups that had formed and unformed vesicles on Day 1, 89.8 ± 7.1 (mean ± SEM, n = 41) and 76.6 ± 7.2 ng mL-1 (n = 27), respectively. On Day 2, in the unformed group, all of the fragments had made vesicles and the IFN-τ increased to 99.4 ± 11.8 ng mL-1. In the extended culture group (n = 10), IFN-τ secretion tended to increase from Day 2 (66.9 ± 14.2 ng mL-1) to Day 8 (166.0 ± 46.7 ng mL-1) (P = 0.06). However, this large amount of IFN-τ on Day 8 significantly decreased from Day 10 (32 ± 4.9 ng mL-1, P &lt; 0.05) to Day 24 (9.2 ± 1.0 ng mL-1, P &lt; 0.05) gradually. The survival rate of these bTVs decreased to 90% (9/10) on Day 10 and then to 60% (6/10) during Days 18 to 22. These results indicate that bTVs cultured for a long term in vitro might decrease IFN-τ secretion. </jats:p

67 Effects of phytohemagglutinin on the culture of isolated bovine blastomeres derived from the 8-cell stage invitro-produced embryos

Monozygotic twin embryos which can efficiently be produced by blastomere separation and aggregation of early cleavage stages of embryos using commercially provided well-of-the-well (WOW) culture dish. Phytohaemagglutinin (PHA) is a plant lectin that binds to and aggregates on the surface of animal cells, but also contains toxicity that causes food poisoning. The present study was conducted to evaluate the toxicity to embryos and the effect to development of isolated blastomeres on PHA-supplemented WOW culture. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and invitro culture (IVC). The tissue culture medium 199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10mgmL−1 bovine serum albumin, and CR1aa medium containing 5% CS were used for each culture step. For the evaluation of PHA toxicity, 89 embryos that developed to the 5-8-cell stage were obtained at Day 2 after insemination. Each embryo was cultured in a droplet of 5 µL/embryo IVC culture medium supplemented with or without PHA. For the evaluation of PHA to development of isolated blastomeres, 111 of 8-cell stage embryos were obtained 48-54h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into single blastomeres by gentle pipetting in IVC medium. Each four blastomeres were formed in the shape of a bunch inside the thin cylinder at the tip of the Pasteur pipette by gentle pipetting. Then, each mass of blastomeres in each 60 masses was cultured individually in 5-µL droplets of IVC medium supplemented with or without PHA on the flat surface of a tissue culture dish. On the other hand, each four blastomeres were introduced into a single conical micro-well each having a diameter and depth of ~287µm and 168µm (Dai Nippon Printing). This culture of blastomeres was performed covered with a droplet of 2.5µL well−1 IVC medium supplemented with or without PHA in each 50 or 52 wells. In all of investigations, PHA was used at 50µgmL−1 (Akagi et al. 2011 J. Reprod. Dev. 57). Statistical analysis was performed using Student's t-test and analysis of variance. The blastocyst formation rate (71.1±2.3% vs. 72.7±1.7%), total cell number (120 vs. 122), and inner cell mass cell number (47 vs. 51) at Day 7 after IVF did not differ between PHA-supplemented and PHA-free group in the toxicity test, respectively. In the blastomere culture, the blastocyst formation rate was very low (10.0±5.9% vs. 5.0±2.9%) regardless of the PHA supplementation in drops on the flat surface of a tissue culture dish. On the other hand, blastocyst formation was improved using the WOW culture dish (24.0±3.6% vs. 40.4±7.6%) but there was no difference with or without PHA supplementation. Although nontoxicity of PHA and efficacy of WOW culture for isolated-aggregated blastomeres were confirmed, no improvement of PHA supplementation on development was observed in this study. Subsequently, experiments on the optimum concentration of PHA for aggregation and development of blastomeres in WOW culture are required. </jats:p

75 DETECTION OF EMBRYONIC DEATH BY MONITORING OVARIAN STEROIDS BALANCE AND ULTRASONOGRAPHY IN JAPANESE BLACK CATTLE

In beef cattle, 20 to 44% embryonic loss occurs during the early stages of pregnancy (Humblot et al. 2001). However, the mechanism of early and late embryonic death is not clear. We investigated occurrence of embryonic death by monitoring ovarian hormones dynamics and checking for the presence of the conceptus after artificial insemination or embryo transfer in beef cattle. Twenty Japanese black females were inseminated (AI) and 12 females were transferred with 1 embryo (ET). Blood samples were collected on Days 21, 24, 28, 38, 48, 58, and 68 post-oestrus (oestrus = Day 0) and then stored at –30°C. Progesterone (P4) and oestradiol (E2) plasma concentrations were analysed using a chemiluminescent immunoassay. On each day of blood sampling, ovaries and presence of a conceptus in the uterus were monitored by ultrasonography. At Day 24 post-oestrus, the presence of the fetus was detected in 8 females (AI, n = 6; ET, n = 2), whereas only 3 females were confirmed to be pregnant at Day 28 and Day 38 post-oestrus. No embryo loss was seen at later stages of pregnancy (Days 48, 58, and 68 post-oestrus). At Day 21 post-oestrus, the conceptus could not be detected by ultrasonography but the E2/P4 ratio provided indication on the pregnancy status of the females that were classified as pregnant (n = 8) or not pregnant (n = 24) at day 24 post-oestrus (1.7 ± 2.2 versus 28.0 ± 34.2 respectively; mean ± s.d.). In the nonpregnant females compared with the pregnant ones at Day 24 post-oestrus, P4 declined below 1 ng mL–1 (0.6 ± 0.2 ng mL–1 v. 8.6 ± 3.9 ng mL–1), whereas E2 blood level remained stable (15.7 ± 21.9 v. 18.1 ± 1.1 pg mL–1). The decrease in P4 levels led to an increase in E2/P4 ratio (1.3 to 37.3 on Day 24). Our study suggests that a large proportion of embryo loss (75%) occurs before Day 24, whereas an additional 16% loss was seen between Day 24 and Day 28 post-oestrus. This embryo loss was shown to be associated with the altered balance of ovarian hormones. </jats:p