Investigation of developmental toxicity and teratogenicity of macrolide antibiotics in cultured rat embryos

Macrolides are considered to be one of the safest anti-infective groups in clinical use, with severe adverse reactions being rare. However, there are limited data about their embryotoxicity and teratogenicity. We aimed to investigate and compare the effects of these agents on embryonic growth and development. Rat embryos were cultured in vitro for 48 h in rat serum. Whole rat serum was used as a culture medium for the control group while different concentrations of spiramycin and azithromycin (1.25-6.25 microg/ml), and clarithromycin (2.5-30 microg/ml) were added to rat serum for the experimental groups. Dose-dependent effects of macrolides on embryonic developmental parameters were compared using morphological methods. Embryos were evaluated for the presence of any malformations. After morphological examination of the embryos, total DNA was extracted from the cells using standard procedures to determine fragmentation of nuclear DNA of embryonic cells. When compared with the control embryos, the macrolides significantly decreased all growth and developmental parameters dose dependently. While clarithromycin was found to cause more developmental toxicity than spiramycin and azithromycin, azitromycin was determined to have more teratogenicity potential. Compared with controls, there was no difference regarding the fragmentation of nuclear DNA of all the agents used. According to these results, when the toxic and teratogenic potential of the used agents compared, because of the lower toxic and teratogenic effects observed with spiramycin, this agent may be preferred for parturients.     

Investigation of Developmental Toxicity and Teratogenicity of Antiemetics on Rat Embryos Cultured In Vitro

In this study, we aimed to investigate and compare the direct toxic and teratogenic effects of dimenhydrinate, metoclopramide and trimethobenzamide HCl, antiemetic drugs on embryonic growth and development in cultured rat embryos. Embryos were explanted on day 9.5 of gestation and cultured. Whole rat serum was used as a culture medium for the control group while different concentrations of dimenhydrinate (2.5–20 μg/ml), metoclopramide (10–50 μg/ml) and trimethobenzamide HCl (25–100 μg/ml) were added to serum for the experimental groups. Effects of antiemetics on embryonic developmental parameters were compared, and embryos were evaluated for the presence of any malformations. Also, the total DNA was extracted from the cells to determine the fragmentation of nuclear DNA of embryonic cells. Compared with the control embryos, the antiemetics significantly decreased all growth and developmental parameters dose dependently. There was no difference regarding the fragmentation of nuclear DNA of the all used agents and controls. Amongst the agents, trimethobenzamide HCl was found to have more toxic and teratogenic potential, and metoclopramide appears to be the least toxic antiemetic and therefore could be more safely used and might be preferred for the treatment of nausea and vomiting in pregnancy.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.     

Anticoagulant effects of low-molecular-weight heparins in healthy cats

BACKGROUND: Low-molecular-weight heparin (LMWH) has potential benefit in cats at risk for thromboembolic disease. However, LMWH pharmacokinetics has not been characterized in the cat. Drug effect with LMWH may be evaluated with analysis of factor Xa inhibition (anti-Xa) or thromboelastography (TEG). HYPOTHESIS: Administration of LMWH at previously recommended dosages and schedules to healthy cats will result in inhibition of factor Xa and hypocoagulable TEG. ANIMALS: In vivo research with heparin was performed in 5 purpose-bred cats. METHODS: In a prospective study with randomized crossover design, heparin or placebo was administered. Treatments were unfractionated heparin (UFH), 250 IU/kg q6h; dalteparin, 100 IU/kg q12h; enoxaparin, 1 mg/kg q12h; or 0.9% saline, 0.25 mL/kg q6h. Each drug was administered for 5 consecutive days followed by a minimum washout of 14 days. Baseline and post-treatment analyses included anti-Xa, TEG, and prothrombin time/activated partial thromboplastin time. RESULTS: Mean anti-Xa activity 4 hours after enoxaparin (0.48 U/mL) approached the human therapeutic target (0.5-1.0 U/mL); however, mean trough anti-Xa activity was below detection limits. Mean anti-Xa activity 4 hours after dalteparin was lower, and only 1 cat attained therapeutic target at a single time point. Cats receiving UFH attained target anti-Xa activity and changes in TEG at trough and 4 hours. CONCLUSIONS: Cats have rapid absorption and elimination kinetics with LMWH therapy. On the basis of pharmacokinetic modeling, cats will require higher dosages and more frequent administration of LMWH to achieve human therapeutic anti-factor Xa activity of 0.5-1 U/mL. Peak anti-Xa activity is predicted at 2 hours after administration of LMWH.     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

Effect of Anti-Basic Fibroblast Growth Factor (Anti-bFGF) on In Vitro Embryonic Development in Rat

In this study, we aimed at the in vitro effects of anti-fibroblast growth factor-2 (anti-FGF-2 or anti-bFGF) on embryo culture in rats. In vitro effects of anti-bFGF on total embryonic development were investigated in 40 rat embryos (which were divided into four groups) (obtained from five pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), and in WRS+ 2.5, 5, and 10 μg/ml anti-bFGF. After 48 h of culturing, the embryos from each group were harvested to be analysed morphologically according to a morphological scoring system and biochemically to obtain the embryo protein content. The morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in WRS+ anti-bFGF had significant embryonic retardation. Mean morphological scores for the embryos grown in WRS, in the presence of 2.5, 5 and 10 μg anti-FGF-2 were 61.4 ± 1.64, 46.3 ± 8.42, 27 ± 2.58 and13.6 ± 0.96 respectively. These results suggest that bFGF is very important for normal embryonic development and rat anti-bFGF neutralizes bFGF effect.     

Teratogenicity of edoferon kappa A, a molecule derived from salicylate, in cultured rat embryos: differences from salicylate and interaction with free oxygen radical scavenging enzymes

The effect of edoferon kappa A (E-KA), a non-specific immunomodulatory and anti-neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA. Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h. E-KA (0.1-12.8 mg/ml) and ASA (0.1-0.6 mg/ml) were added to the whole rat serum. To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E-KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mumol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. E-KA and ASA decreased growth and development in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud). There were no statistically significant differences between the control and embryos grown in the presence of 0.1-0.4 mg/ml E-KA, although the effects of ASA started at a concentration of 0.2 mg/ml. Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5-3.2 mg/ml E-KA and 0.3-0.6 mg/ml ASA were added. When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level. We concluded that E-KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

应用体细胞克隆技术和胚胎移植技术生产克隆水牛

为了解决克隆水牛技术在供核细胞、受体细胞中的选用问题,探索不同条件对移植受孕的影响,完善克隆技术的理论体系。试验以摩拉奶水牛的耳成纤维细胞作为供核细胞,本地水牛卵母细胞为受体胞质进行细胞克隆构建重组胚胎;将发育5～10 d的重组胚胎移植于本地水牛、杂交水牛的子宫内,观察不同发情方式、胚胎类型、胚龄及季节对移植受孕的影响。结果表明:水牛的克隆胚胎妊娠率较低,分别为12.05%、12.04%、13.95%。自然发情和同期发情对克隆胚胎移植受胎率的影响差异不显著;鲜胚胎移植186头,受胎22头,受胎率为11.83%(22/186);冻胚胎移植174头,受胎23头,受胎率为13.22%(23/174)。克隆水牛从出生至12月龄体尺增长明显高于本地水牛;水牛克隆胚胎移植受胎在秋季最好,秋季受胎数占全年的53.33%,显著高于其他季节。     

Addition of Recombinant Human Growth Hormone to In Vitro Maturation Medium of Bovine Oocytes

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.     

Low‐Molecular‐Weight Heparin Dosage in Newborn Foals

Background: Heparin is used in humans as prophylaxis of hypercoagulable states and disseminated intravascular coagulation (DIC). However, babies need a higher heparin dose than do adults. Septic neonate foals are at high risk of hypercoagulable state and DIC, and there is limited objective information about heparin dose for equine neonates. Objective: To assess whether neonate foals require higher dosages of low‐molecular‐weight heparin (LMWH) than adults. Animals: Eighteen healthy and 11 septic neonate foals. Methods: Experimental and clinical studies. Firstly, healthy foals were randomly distributed in 2 groups, 1 receiving 50 IU/kg SC of dalteparin and the 2nd group receiving 100 IU/kg SC of dalteparin, once daily for 3 days. Blood samples were collected before and 3, 6, 27, and 51 hours after the 1st LMWH administration. Plasma antifactor‐Xa activity was measured, together with hemostatic and hematologic parameters used to assess the risk of bleeding. Subsequently, septic foals were treated blindly either with placebo (saline) or 100 IU/kg of dalteparin for 3 days. Plasma antifactor‐Xa activity and other hemostatic parameters were determined before and after treatment. Results: Plasma antifactor‐Xa activity in healthy foals was below prophylactic activity when using the adult dosage (50 IU/kg), whereas prophylactic activities were achieved when using the double dosage (100 IU/kg). No hemorrhagic events and erythrocyte‐related complications were observed with either dosage. In the clinical study, only 4/6 septic foals had plasma antifactor‐Xa activity adequate for prophylaxis. Conclusions and Clinical Importance: Equine neonates require higher dosages of LMWH compared with adults to reach prophylactic heparinemia.     

非繁殖季节胚胎移植受体绵羊妊娠效果影响因素的研究

在非繁殖季节应用CIDR法和Progestagen Sponge法对受体绵羊进行同期发情处理后,进行冷冻胚胎移植,研究不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对冷冻胚胎移植妊娠率的影响,以期为当地实现绵羊胚胎移植产业化提供理论依据和技术参考。结果表明:经同期发情处理后,绵羊在去栓后24~72 h集中发情,在去栓后36~48 h发情最为集中;CIDR法和Progestagen Sponge法的同期发情率分别为91.11%和87.78%(P>0.05),同期发情绵羊移植利用率分别为82.93%和78.48%(P>0.05);单侧卵巢上含有1个和2个黄体其妊娠率分别为46.07%和48.78%(P>0.05);桑椹胚和囊胚的妊娠率分别为49.25%和42.11%(P>0.05);腹腔镜法和手术法的妊娠率分别为45.16%和48.52%(P>0.05);11月和12月进行冷冻胚胎移植的妊娠率分别为50.76%和43.08%(P>0.05),该试验的绵羊冻胚移植妊娠率为46.92%,基本接近国内冻胚移植水平,但低于目前国内羊鲜胚移植妊娠率(50%~60%)。由此可以得出,不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对胚胎移植妊娠率的影响均无显著性差异(P>0.05),大规模群体羊同期发情时,可选价格便宜的Progestagen Sponge法;只要受体绵羊黄体质量好即可移植胚胎;为了减轻受体绵羊的应激反应,降低术后子宫粘连的发病率,可广泛应用腹腔镜法进行胚胎移植;该试验的绵羊冻胚移植妊娠率低于目前国内羊鲜胚移植妊娠率,通过筛选高效低毒的抗冻保护剂,以及提高基层技术人员操作水平,则有望进一步提高绵羊冷冻胚胎移植妊娠率。     

Pharmacokinetics of the low molecular weight heparin dalteparin in cats

Low molecular weight heparin (LMWH) is used as an anticoagulant in cats although only limited pharmacokinetic data are available in this species. The aim of the present study was to establish the pharmacokinetics of dalteparin in cats based on anti-FXa heparin activities. Groups of clinically healthy cats (six animals per treatment) received individual LMWH injections at three different doses intravenously (IV) (25, 50, 100 anti-factor Xa international units [IU anti-FXa]/kg) or subcutaneously (SC) (50, 100, 200 IU anti-FXa/kg). Blood samples were collected before and at various times after injection. Anti-FXa activity was measured with a chromogenic substrate test. Following IV injection, maximum plasma heparin activities (C(max)) were 0.67 ± 0.14, 1.44 ± 0.22 and 2.87 ± 0.38 IU anti-FXa/mL, respectively. The calculated mean half-life (t(?)) was between 39 and 57 min and was not significantly dose-dependent (P=0.139). The volume of distribution (35-39 mL/kg) was almost equivalent to the plasma volume. After SC injection, C(max) values of 0.41 ± 0.10, 0.86 ± 0.17 or 1.91 ± 0.16 IU anti-FXa/mL, respectively, were calculated at 91-110 min post-injection. The t(?) values were between 106 and 122 min and were not significantly influenced by dose (P=0.784). The bioavailability after SC injection was approximately 100%. The high bioavailability of the SC administered LMWH dalteparin in cats was consistent with other species and indicated predictable blood levels. However, the comparatively short t(?) may indicate the necessity of multiple daily injections, which should be verified in clinical trials.     

Progesterone and equine chorionic gonadotropin concentrations around the time of pregnancy loss in mares impregnated by donkeys or stallions

The objective of this study was to compare the incidence of pregnancy loss of mares carrying a mule embryo with that of mares carrying a horse embryo. The possible causes of such mortality were evaluated through serial ultrasonographic evaluations and hormonal monitoring, paying special attention to the role of premature regression of the endometrial cups and its relation to inadequate luteal function.

Twenty-eight mares impregnated by stallions and 19 mares impregnated with donkey semen were evaluated ultrasonographically every week from day 20 to day 150 of pregnancy. The viability of the product was assessed each time, and the diameter of the embryonic vesicle was measured from day 25 to day 60. Blood samples for progesterone and equine chorionic gonadotropin (eCG) determination were taken every week.

Both progesterone and eCG concentrations during normal pregnancies wegre significantly lower in the mares inseminated with donkey semen than in the mares impregnated by stallions (P<.05). In 7 of the mares carrying mule conceptuses to term, the concentrations of eCG remained basal throughout the study. In the other animals from this group, the levels of this hormone did increase but returned to baseline much earlier (on day 77 of pregnancy) than in the mares served by stallions (on day 126 day of pregnancy). There was no significant difference between the growth rate of embryonic vesicles of mares carrying mule embryos and that of mares carrying horse embryos (P>.05).

The incidence of pregnancy loss was significantly higher (P<.05) in mares carrying a mule embryo (36.8 %) than in mares carrying a horse embryo (21.4%); it occurred on average on day 93 of pregnancy in mares carrying mule embryos and on day 43 on mares carrying horse embryos. There was only 1 case in which pregnancy loss was associated with concentrations of both eCG and progesterone that were much lower than the average for the normal pregnancies of the same group, and this was in a mare carrying a horse embryo. The most frequent cause of pregnancy loss was premature luteal regression due to primary luteolysis, as evaluated via peripheral progesterone concentrations. This occurred in 2 mares carrying horse embryos and in 4 mares carrying mule embryos. Three mares carrying mule embryos and 1 carrying a horse embryo had abortions that were not preceded or accompanied by any alteration in progesterone or eCG levels and were thus classified as fetal deaths of non-endocrine origin.

It is concluded that the incidence of pregnancy loss is higher in mares carrying a mule embryo than in mares carrying a horse embryo. However, this is not due to the low progesterone concentrations associated with the premature regression of the endometrial cups that occurs in mares with interspecific pregnancy.     

奶牛新鲜和冷冻胚胎分割移植试验

用简单方法,分割7～8日龄新鲜牛胚胎(1分为2),裸半胚成对移植给66头受体,90天妊检,移植妊娠率为56.1％(37/66)。除6头流产和尚有5头待产外,已有26头受体产犊35头,其中有9对同卵双胎,双胎率为34.6％(9/26),半胚产犊率为29.2％(35/120)。对影响成对半胚移植妊娠率和半胚产犊率的诸多因素如胚胎质量,胚胎在体外停留时间、胚胎发育阶段、受体牛品种、黄体状况等进行了较系统的研究。同时对冷冻胚胎进行了分割试验,移植妊娠率为45.5％(5/11),已产3头犊牛。对快速冷冻和常规冷冻胚胎分割后的移植妊娠率进行了比较,分别为25.0％(1/4)和57.4％(4/7)。     

Developmental capacity and implantation potential of the embryos with multinucleated blastomeres

The presence of multinucleated blastomeres (MNBs) in embryos is associated with poor developmental competence in assisted reproductive technologies. This phenomenon is observed not only in humans but also in other animal species. The purpose of the present study was to investigate the characteristics of embryos with MNBs (MNB embryos) that could be utilized in embryo transfer. The developmental rate of MNB embryos to the blastocyst stage (50.8%) was significantly lower than that of normal embryos (73.3%) (P < 0.05). The clinical pregnancy rates of fresh embryo transfer (ET) using day 2 or day 3 embryos were significantly lower in MNB embryos (5.1%) compared with normal embryos (24.0%) (P < 0.05). In the case of frozen-thawed ET using a single vitrified/warmed blastocyst, however, the clinical pregnancy rate of MNB embryos was close to that of normal embryos (59.1% vs. 52.8%). Thus, the findings of the present study suggest that the frozen-thawed ET of MNB embryos might improve the potential for implantation followed by successful pregnancy.     

The effect of vascular endothelial growth factor on in vitro embryonic heart development in rats

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24-h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 +/- 0.87, 46.6 +/- 1.90 and 52.1 +/- 0.97, somite numbers were 26.5 +/- 0.47, 20.1 +/- 0.63 and 24.4 +/- 0.46, crown-rump lengths were 3 +/- 0.07, 2.4 +/- 0.06 and 2.7 +/- 0.06 mm, and embryo protein contents were 160.5 +/- 7.41, 98.2 +/- 4.81 and 141.1 +/- 10.96 mug per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.     

Effects of ginsenosides on organogenesis and expression of glutathione peroxidase genes in cultured rat embryos

Ginseng has been extensively used around the world for several thousand years as a food or drug. However, recently, several reports have indicated that the organogenesis of cultured embryos is inhibited by treatment with ginsenoside, the principal component of ginseng. In this study, we evaluated the morphological changes of embryos and the gene expression patterns of antioxidant enzymes, 3 types of glutathione peroxidases [GPx; cytosolic (cGPx), plasma (pGPx) and phospholipid hydroperoxide (phGPx) forms], in cultured rat embryos (embryonic days 9.5-11.5) exposed to ginsenosides Rb1, Rg1, Re and Rc at levels of 5, 50 and 100 microg/ml. With regard to total morphological scores, no significant differences were noted in the embryos exposed to all doses of ginsenosides, with the exception of 50 microg/ml of Rc. In the cultured embryos exposed to Rg1, a majority of the developmental parameters were normal, but growth of the hind- and mid- brains and the caudal neural tube was significantly increased compared with that observed in the control group (P<0.05). Furthermore, Rc significantly enhanced the growth of a variety of developmental parameters in the cultured embryos, with the exception of the hindlimbs. According to the results of our semiquantitative RT-PCR analysis, the levels of cGPx and phGPx mRNA in the cultured embryos were unaffected by treatment with the ginsenosides. However, the levels of pGPx mRNA increased significantly in the embryos treated with ginsenosides Re, Rc and Rb1 compared with the control group (P<0.05). These findings indicate that ginsenosides may exert a stimulatory effect on the growth of embryos via differential expression of GPx genes.     

Some Factors Affecting the Rate of Pregnancy after Embryo Transfer Derived from the Brazilian Jumper Horse Breed

This study aimed to evaluate the effects of certain embryo transfer parameters on the pregnancy rate after equine embryo transfer of the Brazilian Jumper Horse breed. The size, embryonic development stage, embryo quality, and synchronization of ovulation between the donor (n = 120) and recipient (n = 420) were evaluated in 396 embryos. Embryo recovery was performed on Day 6-9 after ovulation (Day 0 = day of ovulation). The recipient mares were chosen on the day of embryo recovery, and the transfers were performed that same day. The embryo size (diameter including envelopes; n = 396) ranged from 150 to 3000 μm; 67.1% measured between 400 and 1199 μm. The embryo size (400-1199 μm vs. ≤399 μm); stage of development (n = 396; blastocyst and expanded blastocyst versus compact morula and early blastocyst); quality (n = 396; grade 1 [excellent]), 2 [good], or 3 [poor]); and synchronization of ovulation between the donor and recipient (0, 1, 2, 3, and 4 days versus −1, 5, and 6 days, respectively) all affected pregnancy rate (P < .05). The pregnancy rate did not differ significantly among transfers performed on Days 0, 1, 2, 3, and 4. In conclusion, embryos measuring 400-1199 μm produced higher pregnancy rates in recipients than embryos measuring 150-399 μm, and blastocysts and expanded blastocysts produced pregnancy more efficiently than morulae and early blastocysts. The embryo quality also affected the pregnancy rate. Synchronization of donor and recipient ovulation to Days 0-4 improved the efficiency of embryo transplant.