Preparation of the bitter melon seed extract
230 kg of bitter melon seeds were collected and washed with running and distilled water. The seeds were dehydrated by food dehydrators at 40°C and grounded with a blender. The dried bitter melon seeds were extracted with supercritical carbon dioxide (pressure: 26-27 MPa; temperature 50-53℃). Subsequently, the extract was evaporated by freeze drying for 3 days.
Housing and acclimatization
Throughout the experimental period, female Wistar rats (Rattus norvegicus) rats were housed individually except during the mating period. During acclimatization period, male rats were housed in groups (2 rats/cage). Rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. Enrichment material (wooden chew block) was provided to all rats. A total of 100 male and 120 female rats were obtained from the Jai Research Foundation and acclimated in the experimental room (temperature: 19-22℃; relative humidity: 55-66%; light cycle: 12 h light/dark) for a period of 7 days before the cohabitation.
The rats were fed ad libitum with a standard rodent pellete diet (Certified Teklad Global 14% Protein Rodent Diet, Batch No 2014SC-010621MA, procured from Envigo, Inc., USA) with an unlimited supply of drinking water in polypropylene bottles (capacity 250 mL). Drinking water was filtered through a reverse osmosis (RO) water filtration system.
Procedure for cohabitation and allocation
After the acclimatization period, female rats were cohabitated with untreated male rats (1:1) until the evidence of copulation. Detection of mating was confirmed by the evidence of a copulatory plug in the vagina or by a vaginal lavage for sperm. After confirmation of mating, the female rat was returned to an individual cage (assigned to a group), and the day was designated as day 0 of gestation (GD 0). Mated females were assigned to dose groups by stratified randomization based on GD 0 body weights. Mating of rats was conducted until the requisite number of mated females (25 females/group) were obtained.
Different doses of the test article [control: 0 mg/kg BW; low: 250 mg/kg BW; middle: 500 mg/kg BW; high: 1000 mg/kg BW] prepared in RO water were administered to mated female rats once daily through gavage from the 5th day of gestation (GD 5) to the 19th day of gestation (GD19) at approximately the same time each day in the morning. The dose-volume for the administration was 10 mL/kg body weight. The doses were adjusted according to the most recently recorded body weight. Gavage was performed with a stainless-steel cannula attached to a syringe.
Body weights for the mated female rats were recorded individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation.
Thyroid Hormone Analysis
Serum thyroid hormones were analyzed from all surviving female rats at the terminal sacrifice. T3 and T4 were analyzed with the liquid chromatography tandem-mass spectrometry (LC-MS/MS) technique and TSH was analyzed by the ELISA method.
On day 20 of gestation, all surviving female rats were weighed, euthanised by carbon dioxide asphyxiation, dissected and examined macroscopically. The non-gravid uteri were further examined by staining with 5% ammonium sulphide to confirm non-pregnant status. Ovaries and gravid uteri, including the cervix, were removed, and examined immediately.
Organ weight and histopathology
At the time of terminal sacrifice, the thyroid gland was collected and weighed (post-fixation) from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in all female rats.
Allocation of foetuses for the skeletal and soft tissue evaluation was performed by selecting alternate live foetus. Each litter's first live fetus was allocated to skeletal, and the second live foetus was allocated to soft tissue evaluation.
Definitions of the foetal findings. Malformation: major abnormal structural change considered detrimental to the animal (may also be lethal). Variation: minor abnormal structural change considered to have little or no detrimental effect on the animal; may be transient and may occur relatively frequently in the control population.
All foetuses (100%) were examined externally. External foetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations).
Soft tissue evaluation
Approximately 50% of the foetuses in each litter were subjected to the detailed visceral evaluation by microdissection. The procedure for evaluation of soft tissue was performed with the Staple’s technique.
The remaining approximately 50% of the foetuses in each litter were subjected to the detailed skeletal evaluation after processing and staining with alizarin red. Foetuses were preserved in isopropyl alcohol and glycerin solution. The skeletal evaluation was performed with the Staples and Schnell method.
Good laboratory practice
The study was undertaken in compliance with the guidelines of the “Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International” and “Guidelines for Laboratory Animals Facility” issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The project proposal for the experimentation was approved by the “Institutional Animal Ethics Committee (IAEC)”, JRF.
Data were processed to get mean values and standard deviations (S.D.) with significance between the control and experimental groups. Non-pregnant female rats and female rats with complete resorption were excluded from statistical analysis appropriately.
Kolmogorov-Smirnov test or Shapiro–Wilk test were used to normality check and decide parametric or non-parametric data. Based on the outcome of homogeneity of variance (F-test, Bartlett's test, Levene's test, or Brown–Forsythe test), the significance of parametric data [body weight, body weight change/gain, food consumption, carcass weight, uterine weights, organ weight, male sex ratio/male percent, pre-and postnatal loss (%), live foetus (%), and resorptions (%), ano-genital distance (normalized)] was determined by Student's t-test, Satterthwaite method, or analysis of variance (ANOVA) with Dunnett’s test. The significance of non-parametric data [rreproductive performancee, foetal observations, litter size, number of corpora lutea, number of implants, number of live foetus, number of dead foetus, number of pre-and postnatal losses, number of resorptions] was decided by Kruskal–Wallis test with Dunnett’s test, Mann-Whitney U test, or Chi-square test. The significance was considered p < 0.05.