Experimental animals
Nine adult male and sixty adult female Wistar rats (
Rattus norvegicus albinus) were obtained from the Animal House Facility, Department of Zoology, King Saud University, Riyadh, Saudi Arabia. The initial body weights were 300±20 g for males and 180±20 g for females. Before the initiation of the experiment, animals were acclimatized for one week under standardized laboratory conditions maintained at 22±2°C with a controlled 12 h light:12 h dark cycle. Animals had
ad libitum access to tap water and a standard laboratory pellet diet throughout the experimental period.
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC), King Saud University, Riyadh, Saudi Arabia (Approval No. KSU-SE-23-123; approved on 24 January 2024) and were conducted in accordance with applicable institutional guidelines for the care and use of laboratory animals. The study was conducted and reported in accordance with the ARRIVE 2.0 guidelines and the principles of Replacement, Reduction and Refinement (3Rs). All experimental procedures were designed to minimize animal distress and unnecessary animal use.
Hormonal treatments and ovarian stimulation protocol
Progesterone (P4-300®, 4-pregnane-3,20-dione; Botupharma, Brazil) was used for progesterone priming before gonadotropin stimulation. Equine chorionic gonadotropin (eCG; Syncropart®, Ceva Animal Health, France), which exhibits follicle-stimulating hormone-like activity, was administered to stimulate follicular recruitment and growth, whereas human chorionic gonadotropin (hCG; Chorulon®, Merck Animal Health, USA), which exhibits luteinizing hormone-like activity, was used to induce ovulation.
For progesterone priming, females received subcutaneous progesterone injections at a dose of 5 mg/kg body weight once daily for five consecutive days (
Brown-Grant, 1967). The first eCG injection was administered 24 h after the final progesterone injection. Superovulation was subsequently induced by intraperitoneal administration of eCG followed 48 h later by hCG. Under the standard gonadotropin stimulation protocol, females received 150 IU/kg eCG followed by 75 IU/kg hCG. Under the intensified gonadotropin stimulation protocol, females received fixed doses of 100 IU eCG and 50 IU hCG per animal, irrespective of body weight (
Cornejo-Cortés et al., 2006;
Kon et al., 2014).
For experimental standardization, Day 0 was defined as the day of oocyte collection for
in vitro fertilization (IVF), whereas negative days represented time points preceding IVF. To minimize circadian variation, hormonal administrations were performed at 10:00 a.m. According to the superovulation schedule, eCG was administered on Day -3, followed by hCG on Day -1, 48 h later. Oocyte collection was performed at 6:00 a.m. on Day 0, approximately 20 h after hCG administration, corresponding to the expected time of ovulation
(Aoto et al., 2011).
Experimental design
Sixty adult female rats were randomly allocated to two experimental groups according to progesterone priming before
in vitro embryo production. Females in the first group received gonadotropin stimulation without progesterone priming, whereas females in the second group received progesterone priming before gonadotropin stimulation. Within each group, females were further assigned to either the standard or intensified gonadotropin stimulation protocol described above.
A 2 × 2 factorial experimental design was used to evaluate the effects of progesterone priming and gonadotropin stimulation intensity. Accordingly, four treatment subgroups were established: CTRL (standard gonadotropin stimulation), OVD (intensified gonadotropin stimulation), P-CTRL (progesterone priming with standard gonadotropin stimulation) and P-OVD (progesterone priming with intensified gonadotropin stimulation). Each treatment group comprised 15 females distributed equally across three independent experimental replicates, with five females per treatment group in each replicate (20 females per replicate).
The experimental groups, hormonal treatment protocols and administration timing are summarized in Table 1.
In vitro embryo production (IVEP)
Media preparation
Modified Human Tubal Fluid (mHTF) medium was used for gamete collection and fertilization procedures. The medium consisted of 94.6 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl
2, 1.19 mM KH
2PO
4, 1.19 mM MgSO
4, 25.07 mM NaHCO
3, 21.58 mM sodium lactate, 0.5 mM sodium pyruvate, 5.56 mM glucose, 4.0 mg/mL bovine serum albumin (BSA), 50 µg/mL streptomycin sulfate and 75 µg/mL potassium penicillin G
(Kito et al., 2004).
Modified potassium simplex optimized medium (mKSOM) was used for embryo culture following fertilization. The medium contained 95 mM NaCl, 5 mM KCl, 1.71 mM CaCl
2·2H
2O, 0.2 mM MgSO
4·7H
2O, 25.0 mM NaHCO
3, 10.0 mM sodium lactate, 0.2 mM sodium pyruvate, 0.2 mM glucose, 0.01 mM EDTA, 1× minimum essential amino acid solution, 1 mM glutamine, 1× MEM non-essential amino acid solution, 1 mM taurine, 1 mM glycine, 1 mM glutamate, 1 mM alanine and 1 mg/mL BSA
(Men et al., 2023).
Oocyte collection
Females were euthanized by CO
2 inhalation approximately 20 h after hCG administration. Both oviductal ampullae were carefully dissected and transferred into mHTF droplets under paraffin oil. A small incision was made in the ampullary wall using an insulin syringe needle to release the cumulus-oocyte complexes (COCs) into the medium
(Aoto et al., 2011; Men et al., 2023; Oh et al., 1998).
Following recovery, COCs were collected and counted under a stereomicroscope before fertilization. The total number of oocytes recovered from each female was recorded as a measure of ovarian response.
Oocytes from each female were collected, counted, fertilized and cultured separately, without pooling between females or treatment groups. Oocyte yield and subsequent embryo development were recorded individually for each female, which served as the experimental unit for all outcome measurements.
Sperm collection and in vitro fertilization
Adult male rats were euthanized by CO
2 inhalation on the morning of the experiment. The cauda epididymides were carefully excised, trimmed of surrounding fat and connective tissue and transferred into mHTF droplets under paraffin oil. Small incisions were made in the cauda epididymides using micro-spring scissors to release spermatozoa into the fertilization medium
(Aoto et al., 2011; Men et al., 2023; Miyoshi et al., 1997). In each of the three independent experimental replicates, spermatozoa from three different males were pooled into a single sample. The same pooled sperm suspension was used to inseminate oocytes from all four treatment groups (CTRL, OVD, P-CTRL and P-OVD) within each replicate, ensuring a common sperm source across treatments.
Following sperm release, the sperm concentration was determined and adjusted to a final concentration of 1 × 10
6 sperm/mL in the COC-containing fertilization droplets. Before insemination, the sperm suspension was incubated for approximately 60 min at 37°C under 5% CO
2 to allow capacitation. The fertilization dishes were then incubated at 37°C under 5% CO
2 for approximately 12 h
(Aoto et al., 2011; Men et al., 2023; Miyoshi et al., 1997).
Embryo culture and developmental assessment
Following the fertilization period, all oocytes were transferred to mHTF medium supplemented with 1% hyaluronidase and the surrounding cumulus cells were removed using glass Pasteur pipettes. Denuded oocytes were washed twice in fresh mKSOM medium
(Aoto et al., 2011; Men et al., 2023) and then cultured in mKSOM medium at 37°C under 5% CO
2 for 24 h. Early embryonic development was assessed by recording the number of embryos that reached the two-cell stage.
Two-cell embryo yield was calculated using the following equation:
