Morphological features of adult rats of IS/Kyo and IS-Tlk/Kyo strains with lumbar and caudal vertebral anomalies.

IS-Tlk/Kyo, a mutant derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to the congenital lumbar vertebral anomaly. Homozygotes of Tlk dominant gene are known to die during embryonic development. We previously reported the morphological features of the skeleton in IS/Kyo and IS-Tlk/Kyo fetuses and of the heart in IS/Kyo fetuses [19]. This study was conducted to clarify the morphological features of the skeleton in both adult rats and of the heart in adult IS/Kyo rats. Ventricular septal defect (VSD) was observed in 3 out of 10 IS/Kyo rats. Neither splitting of lumbar vertebra and supernumerary rib (in both strains) nor fused or absent caudal cartilage (in IS-Tlk/Kyo strain) was detected in adult rats. Fusion of lumbar vertebrae was observed in almost all specimens together with lumbarization of sacral vertebrae in a few specimens in both adult rats as well as fusion of sacral and caudal vertebrae only in adult IS-Tlk/Kyo rats. In addition, a severe reduction in the ossified sacral and caudal vertebrae was noted in adult IS-Tlk/Kyo rats (mean number: 20.6) and IS/Kyo rats (31.8), and the difference was similar to that in the length of sacral and caudal vertebrae. These results suggest that the Tlk gene may be involved in both the congenital and acquired abnormal formation of the lower vertebral centra as well as the persistent occurrence of VSD by the background gene in IS/Kyo strain.

Polyphenols extracted from black tea (Camellia sinensis) residue by hot-compressed water and their inhibitory effect on pancreatic lipase in vitro.

Polyphenols, retained in black tea wastes following the commercial production of tea beverages, represent an underutilized resource. The purpose of this study was to investigate the potential use of hot-compressed water (HCW) for the extraction of pancreatic lipase-inhibiting polyphenols from black tea residues. Black tea residues were treated with HCW at 10 °C intervals, from 100 to 200 °C. The resulting extracts were analyzed using high-performance liquid chromatography-mass spectrometry and assayed to determine their inhibitory effect on pancreatic lipase activity in vitro. Four theaflavins (TF), 5 catechins, 2 quercetin glycosides, quinic acid, gallic acid, and caffeine were identified. The total polyphenol content of extracts increased with increasing temperature but lipase inhibitors (TF, theaflavin 3-O-gallate, theaflavin 3'-O-gallate, theaflavin 3,3'-O-gallate, epigallocatechin gallate, and epicatechin gallate) decreased over 150 °C. All extracts inhibited pancreatic lipase but extracts obtained at 100 to 140 °C showed the greatest lipase inhibition (IC(50) s of 0.9 to 1.3 µg/mL), consistent with the optimal extraction of TFs and catechins except catechin by HCW between 130 and 150 °C. HCW can be used to extract pancreatic lipase-inhibiting polyphenols from black tea waste. These extracts have potential uses, as dietary supplements and medications, for the prevention and treatment of obesity.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats.

This study measured blood parameters, particularly those related to coagulation, and alterations in the expression levels of blood-coagulation-related genes in lactating Sprague-Dawley rats. The day of delivery was designated as lactation day 0 (LD 0). On the day after delivery (LD 1), prothrombin time and overall activity of vitamin-K-dependent coagulation factors were decreased, whereas fibrinogen contents, platelet counts and antithrombin III concentrations were increased as compared with those in nonpregnant rats. In addition, hepatic expression of blood-coagulation-related genes in the liver was increased at LD 0 as compared with that in nonpregnant rats. These changes may be physiologic responses to prevent prolonged bleeding at delivery. Except for fibrinogen content, which remained elevated, the described changes returned to baseline on and after LD 7. Activities of AST, ALT, and ALP were increased on LD 7, 14, and 21 as compared with nonpregnant rats. In contrast, total protein, albumin, Cl, and Ca were consistently lower on LD 7, 14, or 21 as compared with levels in nonpregnant rats. These results provide background data for evaluation of nursing rats.

Gastric hairballs in rabbits: significance in developmental toxicity study.

We surveyed 1053 pregnant rabbits of the Kbl:NZW strain collected from 27 developmental toxicity studies to reveal the prevalence and significance of gastric hairballs. The incidence of hairballs was 2/525 (0.4%) in the control group and 17/528 (3.2%) in the high dose group. In the high dose group, 16 dams resulted in abortion or death. In addition, decreases in body weight and food consumption were more severe in dams with hairballs than in their group-mates without hairballs.

Morphological features of mutant rat, IS-Tlk/Kyo, fetuses with caudal vertebral anomalies.

IS-Tlk/Kyo, a rat mutant strain derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to a congenital anomaly of the lumbar vertebrae that is a hallmark of IS/Kyo rats. Homozygotes (Tlk/Tlk) of Tlk dominant gene are known to die during embryonic development. The present report deals with the morphological features of heterozygous IS-Tlk/Kyo rat fetuses in comparison with those of IS/Kyo rat fetuses. One of the morphological features was a high incidence of tail vertebral anomalies in IS-Tlk rats (81.6% versus 0% in IS/Kyo rats). Significantly low values in number of live fetuses and ossified 5th sternebra and sacral and caudal vertebrae were observed in IS-Tlk/Kyo rats compared with those in IS/Kyo rats as well as a low incidence of fetuses with ventral septal defects in IS-Tlk/Kyo (0% versus 54.4% in IS rats). These results suggest that the Tlk gene may be involved in the formation of the vertebral centra and the ventral septum when it expresses on the genetic background of the IS rat.

Effects of restricted feeding on fetal and placental development in pregnant rabbits.

In our previous study on the effects of restricted feeding on pregnant rabbits (Matsuoka et al., 2009), animals given 20 g/day of diet on and after gestation day 6 (GD 6) showed significant changes in blood coagulation-related parameters suggesting a tendency to bleed and a decrease in serum concentration of progesterone, an important factor to maintain pregnancy, on GD 22, and a half of them showed serum progesterone concentrations less than 4.0 ng/ml which resulted in abortions on and after GD 23. In the present study, the effects of restricted feeding of 20 g/day from GD 6 to GD 22 on embryo-fetal and placental development on GD 23 as well as on blood coagulation-related parameters and serum progesterone concentrations on GD 22 were examined in pregnant rabbits. As compared with the non-restricted feeding (Not-treated, NT) group, the restricted feeding (RF) group showed lower values of platelets, fibrinogen, activated partial thromboplastin time (APTT) and antithrombin III (ATIII) and a longer prothrombin time (PT), reflecting an inhibition of blood coagulation, and a decrease in serum progesterone concentration on GD 22. Cesarean section performed on GD 23 revealed that the RF group showed a tendency towards an increase in the embryo-fetal death index and lower body weights and placental volumes compared with the NT group. Histological examination of the placenta in the RF group revealed that the labyrinth zone was thin and many glycogen-containing cells still remained in the basal zone, suggesting a delay in placental growth.

Prenatal developmental toxicity study of ethyl tertiary-butyl ether in rabbits.

Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, the prenatal developmental toxicity of ETBE was determined in rabbits. New Zealand white rabbits were given ETBE by gavage at 100, 300, or 1,000 mg/kg/day on gestational days (GDs) 6-27, and the pregnancy outcome was determined on GD 28. Neither death nor abortion occurred in the pregnant rabbits at any dose. Slightly and significantly suppressed maternal body-weight gain and transiently decreased maternal food consumption were found at 1,000 mg/kg/day during the administration period. At this dose, no changes in clinical or macroscopic finding were noted in dams. No treatment-related changes were observed in any dam treated at 300 mg/kg/day or less. There was no significant effect of ETBE on the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, incidences of pre- and postimplantation loss, viability of fetuses, fetal body weight, sex ratio of fetuses, or weights of gravid uteri. No significant difference was detected in the incidences of fetuses with malformations or variations between the ETBE-treated and control groups. Also, no adverse effects on the progress of ossification were noted in fetuses of dams given ETBE. Based on these findings, it is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Changes in blood parameters in pregnant Japanese White rabbits.

Changes in blood parameters, especially those related to blood coagulations, were examined on gestational days (GDs) 6, 18 and 28 in Japanese White rabbits. As compared with the non-pregnant group, the following changes were observed in the pregnant group. For blood coagulation-related parameters, fibrinogen, platelets and antithrombin time III (ATIII) was significantly increased on GD 18 and/or GD 28, and activated partial thromboplastin time (APTT) was significantly prolonged on GDs 18 and 28. For the other hematological and blood chemical parameters, hemoglobin and hematocrit values were significantly decreased on GDs 18 and 28. Reticulocyte counts significantly and prominently increased on GD 18 and then decreased to significantly lower levels than those in the non-pregnant group on GD 28. Eosinophils and basophil showed significant decreases on GD 18 and/or GD 28. The serum progesterone concentration reached its highest level on GD 18. Total protein (TP), albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), cholesterol, calcium, glucose and blood urea nitrogen were significantly decreased on GD 18 and/or GD 28. These results were almost similar to those in the pregnant New Zealand White rabbits (Mizoguchi et al., 2010).

Changes in blood parameters in New Zealand White rabbits during pregnancy.

Changes in clinical pathology parameters, particularly those related to blood coagulation, were examined throughout the gestation period in New Zealand White rabbits. As compared with the non-pregnant group, the following major changes were observed in the pregnant group. For blood coagulation-related parameters, platelets increased progressively and fibrinogen increased slightly from organogenesis, prothrombin time was significantly prolonged during organogenesis and shortened in the late fetal growth stage, activated partial thromboplastin time was significantly prolonged during the fetal growth stage, and antithrombin III increased during and after late organogenesis. Such changes in blood coagulation-related parameters during the later stages of gestation seem to be physiological responses in preparation for protecting against excessive haemorrhage or haemostasis at parturition. For the other haematological and blood chemical parameters as well as progesterone, red blood cell counts, haemoglobin and haematocrit began to decrease during organogenesis and continued to decrease thereafter. Reticulocyte counts significantly increased during organogenesis and decreased thereafter. White blood cell parameters, except for neutrophils, showed significant decreases during the fetal growth stage. Serum progesterone concentration reached its highest level early in organogenesis and decreased thereafter. Total protein, albumin, glucose, cholesterol, calcium, blood urea nitrogen and creatinine decreased significantly during the middle and/or late periods of gestation. In conclusion, the data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rabbits, and pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation.

Changes in blood coagulation-related parameters in phenobarbital-treated rabbits.

The effects of repeated administration of phenobarbital (PB) on blood coagulation time were examined using male Japanese white SPF rabbits, which are widely used for toxicological studies. PB was administered to the rabbits by oral gavage for 2 weeks, at dose levels of 0, 12.5, 25 and 50 mg/kg/day. Blood was collected on Days 8 and 14 after each day's dosing to perform blood coagulation examination. The liver was excised, weighed and examined histopathologically. Activated partial thromboplastin time (APTT) was prolonged at dose levels of 12.5 mg/kg/day or more and Thrombotest (TBT) was prolonged at 50 mg/kg/day on Day 8. APTT was prolonged at dose levels of 12.5 mg/kg/day or more, TBT was prolonged at 25 mg/kg/day or more and factor IX activity decreased at 50 mg/kg/day on Day 14. At pathological examination, liver weight increased at dose levels of 25 mg/kg/day or more, and a ground-glass appearance of the hepatocytes was observed in the central and middle parts of lobules at 12.5 mg/kg/day or more. However, changes in factor VII or X activity or prolongation of prothrombin time (PT) were not observed. Therefore, prolongation of blood coagulation time by PB administration in rabbits was considered to be due to PB's effect on the endogenous pathway alone. Moreover, an increase in anti-thrombin III (ATIII) concentration was noted at 50 mg/kg/day; however, no change was noted at dose levels of 25 mg/kg/day or less. This suggests that the contribution of ATIII to the PB-induced prolongation of coagulation time in rabbits was small.

Effects of restricted feeding on blood parameters in pregnant rabbits.

The effects of restricted feeding (20 g/day from gestational day (GD) 6 to 28) on pregnancy outcome and blood parameters were examined in pregnant rabbits. As compared with the group which was allowed free access to diet throughout the gestational period (NT group), the group subjected to restricted feeding (R group) showed significantly lower values in many parameters such as total protein, albumin and triglyceride on GDs 22 and 28, reflecting low nutritive conditions. In addition, there were significant changes in blood coagulation-related parameters, suggesting an imbalance between coagulation and anti-coagulation factors. Moreover, abortions occurred in about half of the animals of the R group between GDs 23 and 27. When aborted rabbits were compared with those which could maintain pregnancy under restricted feeding, total protein, albumin, platelets and antithrombin III values and especially blood progesterone concentration were significantly lower in aborted rabbits on GD 22, prior to occurrence of abortion. These results suggested that abortions due to restricted feeding might be brought about by lower nutritive conditions, an imbalance of blood coagulation-related factors and lower blood progesterone concentrations.

Characteristics of Kbl:Dutch rabbits in prenatal development in comparison with Kbl:New Zealand white rabbits.

Prenatal development of Kbl:Dutch rabbits was studied in comparison with Kbl:New Zealand white rabbits. Significantly accelerated ossification of the 5th and 6th sternebrae and a low incidence of fetuses with a 13th rib were characteristic features in the prenatal development of Kbl:Dutch rabbits in comparison with Kbl:New Zealand white rabbits. These characteristics were largely consistent with earlier studies of Dutch rabbits from different suppliers and are notable when Kbl:Dutch rabbits are used for the evaluation of skeletal ossification.

Effect of lactulose on calcium and magnesium absorption: a study using stable isotopes in adult men.

To evaluate the effect of lactulose on calcium (Ca) and magnesium (Mg) absorption, we performed a clinical trial with a double-blind, randomized, crossover design in 24 healthy adult male volunteers. The absorptions of Ca and Mg were evaluated by a single-labeling method using stable isotopes. The test foods, containing lactulose at a dose of 0 g (placebo), 2 g (low-dose), or 4 g (high-dose) together with 300 mg of Ca containing 20 mg of 44Ca, and 150 mg of Mg containing 28 mg of 25Mg, were administered orally. Urine samples were collected for 8 h after the ingestion of the test food. The ratios of stable isotopes in urine (44Ca/40Ca and 25Mg/24Mg) were measured by ICP-MS (inductively coupled plasma-mass spectrometry). The urinary stable-isotopes ratios (44Ca/40Ca and 25Mg/24Mg) increased with lactulose dosage. Significant differences were observed in the Ca ratio between placebo and high-dose lactulose (p<0.01), and in the Mg ratio between placebo and low-dose lactulose and between placebo and high-dose lactulose (p<0.01). Lactulose ingestion did not change the levels of bone-resorption markers (type I collagen cross-linked N-telopeptide and deoxypyridinoline) in urine. The test foods did not cause any side effects. This study demonstrates that lactulose enhances the absorptions of Ca and Mg in adult men.

An investigation on restraining effects and acclimation to protecting jackets in pregnant rabbits -wearing from pre-mating through fetal organogenesis-.

The purpose of the present investigation is to evaluate the restraining effects of protecting jackets attached from the pre-mating period and thereafter up to completion of the fetal organogenesis in rabbits. Forty (40) Kbl:NZW mated rabbits 6 to 10 months of age were divided into 2 groups of 20 animals each. Animals in the treatment group (hereinafter referred to as JK group) were dressed in protecting jackets from Day -9 prior to mating (hereinafter referred to as GD -9) to GD 19 and another group of animals without protecting jackets was used as a control. The major restraining effects noted were decreases in body weight gain and food consumption, which were marked in the first 9 days of jacket-attachment during the pre-mating period. The decrease in body weight was still apparent over the whole gestation period. However, the mean body weight of the JK group finally reached the baseline level of the study at around GD 11. These findings demonstrated that the restraining effects in the present study persisted longer than those in the previous study, in which protecting jackets were used from GD 0 to GD 19 or GD 6 to GD 19. The fertility function and cesarean section findings were comparable to those in the control group. It may be concluded that the use of protecting jackets from GD -9 to GD 19 induced more prolonged effects on body weight loss than in previous findings where the jackets were attached from GD 0 to GD 19 or GD 6 to GD 19 and the time of starting the acclimation with the jackets appears to play a great role in reducing body weight loss.

Effects of stage and degree of restricted feeding on pregnancy outcome in rabbits.

This study's aim is to investigate the relationship between stage and degree of restricted feeding during the gestation period and occurrence of abortion, premature birth or fetal damage in rabbits. The study was composed of 5 groups of pregnant Kbl:NZW rabbits that consisted of 8 animals each. These groups were subjected to restricted feeding in the following ways: (A) control group, free access to food, (B) 60 g per day from gestational days (GD) 6 to 18 (middle period), (C) 20 g per day from GD 6 to 18, (D) 20 g per day from GD 19 to 28 (post-middle period), and (E) 20 g per day from GD 6 to 28 (middle and post-middle periods). Even though all dams in Groups A, B and C went to full term, abortion or premature birth occurred to 2/8 and 8/8 dams in Groups D and E, respectively. Fetal lethality increased in Group C, which was subjected to restricted feeding at 20 g/head/day in the middle period. Slight inhibition of fetal growth was recorded only in Group D, which was subjected to restricted feeding in the post-middle period. Restricted feeding at 20 g/head/day in the middle period induced no abortion or premature birth, but increased fetal lethality that in the middle and post-middle periods resulted in abortion or premature birth of all dams, and that in the post-middle period resulted in abortion or premature birth at low incidence and slightly inhibited fetal growth. These results demonstrated that the post-middle period is vulnerable to effects of reduced food consumption in pregnant rabbits.

Restraining effects of and habituation to protecting jackets in pregnant rabbits.

The present study was undertaken to elucidate restraint effects of protecting jackets that are used to protect against biting the sites of percutaneous application in pregnant rabbits. Animals wore protecting jackets from gestational Day (GD) 0 to 19 (GD 0 group) or from GD 6 to 19 (GD 6 group). Major restraint effects were decreases in body weight gain and food consumption, which were marked during the first 2 to 13 days in jackets, and diminished thereafter. Therefore, the animals in the GD 0 group had almost recovered from the effects on GD 16, but those in the GD 6 group were still under the effects of restraint on GD 19. The above findings indicate that early start of habituation to protection jackets will provide better results in practical reproduction studies using percutaneous application in rabbits.