Abstract
Background/Aim: Domestic pigs have become increasingly important models in translational research; however, their large size presents logistical and ethical challenges. Microminipigs offer a practical alternative for long-term studies. This study aimed to develop an effective superovulation protocol using a step-down follicle-stimulating hormone (FSH) regimen to improve zygote collection in microminipigs.
Materials and Methods: In experiment 1, four female microminipigs with regular estrous cycles were used in both the control and treatment conditions in a crossover design. Treatment group was received prostaglandin F2α (PGF2α), followed by step-down FSH, and human chorionic gonadotropin (hCG). Follicle number, area, and diameter were monitored by ultrasonography from day −6 to day +1 of estrus. In experiment 2, three female microminipigs were used. Zygotes were retrieved at day 1 by oviduct flushing.
Results: On day 0, the number of follicles was higher in the treatment group (93.0±7.74) compared to the control (46.8±5.01). Significant differences were observed on days −1 and 0, while by day+1, the number of follicles decreased in both groups. Follicle area was significantly larger in the treatment than in the control group (0.81±0.03 cm2 vs. 0.63±0.04 cm2) on day −2, with no significant differences detected on days 0 and +1. Follicle diameter was also significantly greater in the treatment group compared to the control (3.4±0.1 mm vs. 2.7±0.1 mm) on day −2, while no significant differences were found on days −1, 0, and +1. In experiment 2, an average of 14.7 zygotes per animal were recovered. The cleavage and blastocyst formation rates were 62.8% and 59.4%, respectively.
Conclusion: The step-down FSH protocol effectively enhanced ovarian response and embryo yield in microminipigs, marking a foundational step toward the efficient reproductive engineering for this animal model that may contribute to the advancement of translational research.
Introduction
Pigs have attracted increasing attention as animal models in translational and nonclinical research owing to their anatomical, immunological, genetic, and physiological similarities to humans (1-3). Recent advances in biotechnology have enabled the development of transgenic pigs that can model various human diseases, such as diabetes mellitus (4), further promoting their use in biomedical research. In addition, genetically engineered pigs lacking specific antigens are being investigated for xenotransplantation, which is gaining recognition as a promising approach to address the critical shortage of human donor organs in transplantation medicine (5).
Although pigs are considered one of the most suitable species for translational research, they are not without limitations. Domestic pigs grow rapidly and can weigh more than 200 kg in adulthood, making them difficult to handle and maintain. To overcome these size-related challenges, several miniature pig breeds have been developed for use in biomedical research (6). Microminipigs developed by Fuji Micra Inc. are among the smallest breeds in the world, reaching an adult body weight of approximately 30 kg, and are particularly suitable for translational research owing to their manageable size and physiological characteristics (7). These small pigs, which do not exhibit the rapid growth observed in livestock pigs, can be maintained for extended periods and allow longitudinal monitoring using medical devices such as computed tomography (CT) and magnetic resonance imaging (MRI) (8, 9). Therefore, they are considered promising animal models for translational research.
Genetic modifications play a pivotal role in generating animal disease models (10). In recent years, a widely adopted method for gene introduction in pigs has involved the direct electroporation of zygotes (11, 12). In particular, the introduction of CRISPR/Cas9 has enabled the generation of pigs with various gene knockouts (13). In domestic pigs, genome editing is typically performed in oocytes collected from slaughterhouse-derived ovaries. However, in miniature pigs, zygotes must be collected directly from these animals. Therefore, the efficient collection of zygotes derived from miniature pigs is essential for the successful production of genetically modified miniature pig models.
In microminipigs, the average litter size is approximately five piglets (14), and the timing of ovulation can be accurately determined using regular ultrasonography. This enables the collection of oocytes or fertilized embryos at an optimal stage of development. However, a significant section of the unoccupied uterus on day 12 prevents the continuation of pregnancy, regardless of the number of embryos present in the occupied section (15). In domestic pigs, the transfer of 16-20 embryos is considered optimal for achieving normal pregnancy rates, suggesting that is in microminipigs, approximately 10 embryos, roughly twice the normal average litter size, may be required to achieve comparable success rates (16). In addition, although synchronizing ovulation in multiple microminipigs may improve the likelihood of generating genetically modified offspring, maintaining and utilizing large numbers of animals is inefficient and raises ethical concerns regarding animal welfare. Therefore, the development of an effective superovulation protocol in microminipigs is a critical step toward the more efficient production of genetically engineered models to advance translational biomedical research.
Two experiments were conducted in this study. The first aimed to establish a superovulation protocol using a step-down follicle stimulating hormone (FSH) administration method, which has been reported to be effective in miniature pigs (17). In contrast to long-acting eCG-based protocols used in some miniature pig studies, we used FSH to achieve more precise control over follicular stimulation (18, 19). To our knowledge, this FSH step-down protocol has not been previously reported in microminipigs, and it was developed from similar treatments used in other livestock species (20, 21). To evaluate its efficacy, ovarian responses were monitored throughout the treatment period using ultrasonography. The second experiment focused on collecting zygotes after superovulation and evaluating their quality by observing early embryonic development in vitro.
Materials and Methods
Ethical approval. The protocols for animal use were approved by the Animal Care and Use Committee of Gifu University (#2020-089) and were conducted in accordance with the university’s guidelines for animal research and welfare. All pigs were cared for by a veterinarian throughout the experiment.
Animal husbandry. The animals were housed in a temperature-controlled environment (21-27°C) with a relative humidity of 30-80% and a 12-h light/dark cycle. Each pig was fed Multilack (Chubu Shiryo Co., Ltd., Nagoya, Japan) once daily, according to the supplier’s instructions.
Microminipigs for experiment 1. Following preliminary experiments aimed at establishing a superovulation protocol (data not shown), four female microminipigs (pigs A-D), aged 1-2 years and weighing 16-20 kg, were purchased from Fuji Micra Inc. (Fujinomiya, Shizuoka, Japan). To minimize inter-individual variation, the same microminipigs (A-D) were used for both the control and superovulation groups. Prior to each experiment (at least two estrous cycles were observed over an interval of two to three months. During this period, the pigs displayed normal estrous behavior, and the estrous cycle, as confirmed by genital observation and backpressure testing, had a median (range) of 21 days (21-23 days), consistent with a previous report.
Superovulation protocol. Preliminary trials based on previous reports (8, 17, 22) were conducted to optimize the superovulation protocol in microminipigs prior to this study (unpublished data). The onset of standing estrus in response to a boar was designated as day 0, and the superovulation protocol was designed accordingly. On day −6, 1.5 mg of PGF2α was administered twice. This was followed by step-down administration of FSH every 12 h, using the following schedule: 1.2 AU on day −5, 0.8 AU on day −4, and 0.6 AU on day −3. Finally, 400 IU of hCG was administered 24 h after the last FSH treatment.
Observation and evaluation of follicular development. Estrous behavior was observed by the female gilt’s standing reaction to the boar. Ovarian activity was monitored daily from day −6 to day 1 using ultrasonography. For ovarian monitoring, the pigs were sedated via intramuscular administration of 0.015 mg/kg medetomidine (Dorbene vet; Kyoritsu Seiyaku Corporation, Tokyo, Japan), 0.15 mg/kg midazolam (Dormicum injection 10 mg; Astellas, Tokyo, Japan), and 0.12 mg/kg butorphanol (Vetorphale; Meiji Seika Pharma Co., Tokyo, Japan) (9). After the ultrasound examination, the pigs were promptly awakened by administering 0.064 mg/kg of atipamezole (Kyoritsu Seiyaku). Throughout the trial, the pigs were carefully monitored by the veterinarians to ensure they were healthy.
To assess follicular development, a portable ultrasound system (ARIETTA Prologue; FUJIFILM, Tokyo, Japan) was used to monitor hormonal responses on days days −6, −2, −1, 0, and 1. Following sedation, the pigs were placed in a lateral recumbent position, and an ultrasound probe was applied to the abdominal surface to capture video recordings of the ovarian dynamics from the above duration, from day −6 to day 1. Using OsiriX MD (version 14.1), the number of follicles, follicle diameter, and follicular area were manually acquired from 800 to 1,400 sequential ovarian images in DICOM format obtained from the video recordings. Follicular detection and measurements were performed independently by two examiners to ensure accuracy.
Microminipigs for experiment 2. Three microminipigs (pigs A-C) were used in experiment 2. Pigs A, B, and C were 42, 72, and 38 months old, respectively, with body weights of 32.8, 40.0, and 40.0 kg, respectively. The pigs were raised under the same conditions as those used in experiment 1.
Zygote collection. The estrous cycles of the microminipigs were monitored, and mating was performed at the optimal time with a microminipig boar on day 0. On day 1, laparotomy was performed under isoflurane anesthesia (DS Pharma Animal Health, Osaka, Japan), and fertilized embryos were collected via oviductal flushing with phosphate-buffered saline (PBS) preheated to 37°C. The number of corpora hemorrhagicum (CH), used as indicators of ovulation, was also recorded.
The collected zygotes were initially cultured in an incubator maintained at 5.0% CO2, 5.0% O2, 39°C, and 100% humidity. From days 1 to 3, the first culture medium used was PZM-5 (Research Institute for Functional Peptides, Yamagata, Japan). Subsequently, from days 4 to 6, the embryos were transferred to the second culture medium, PBM (Research Institute for Functional Peptides). Cleavage and blastocyst formation rates were assessed on days 2-6.
Statistical analysis. The analysis was conducted using IBM SPSS Statistics software (version 29.0.20, IBM, Armonk, NY, USA), employing one-way analysis of variance (ANOVA) to compare the means between the treatment and control groups. A follow-up Welch’s t-test was performed for post-hoc analysis to account for the potential heterogeneity of variance among the groups.
Results
Experiment 1: Follicular development at superovulation treatment. Superovulation treatment with step-down FSH administration was performed, and the follicular area, diameter, and number were measured using ultrasound on days −6, −2, −1, 0, and 1 to assess follicular development (Figure 1).
Follicular changes associated with superovulation treatment. Follicles were shown on ultrasound DICOM images using OsiriX MD software. Arrowheads indicate individual follicles. On day −6, the ovaries in both groups were difficult to identify, with few detectable follicles. However, by day −2, multiple follicles had developed, making the ovaries clearly distinguishable in both groups. From day −2 to day 0, the treatment group exhibited a significantly higher number of follicles than the control group. By day 1, most of these follicles had disappeared, suggesting that ovulation had occurred.
The number of follicles in the treatment group increased significantly on days −2, −1, and 0. On day −6, prior to the initiation of the superovulation treatment, the follicle counts were 18.00±5.20 (Mean±SEM) in the treatment group and 23.25±1.44 in the control group, with no statistically significant difference observed between the two groups (p=0.393) (Figure 2). On day −2, the number of follicles in the treatment group was 79.75±11.05, whereas that in the control group was 37.50±6.89, showing a significant increase in the treatment group (p=0.023). On day −1, follicle counts further increased to 89.0±9.63 in the treatment group compared to 38.0±6.82 in the control group (p=0.006). On the day of estrus, the number of follicles in the treatment group was 93.0±7.74, while that in the control group was 46.8±5.01, showing a significant increase in the treatment group (p=0.004). Following estrus on day 1, the number of follicles decreased in both groups (33.3±3.92 in the treatment group and 24.0±5.21 in the control group), suggesting that ovulation had occurred. No statistically significant differences were observed between the groups on day1 (p=0.210).
Ovarian response to superovulation treatment. A significant increase in the number of follicles was observed on day −2 after FSH administration. The decrease in follicle number from day 0 to day 1 suggests that ovulation likely occurred during this period (A). In response to hormonal treatment, both follicle diameter (B) and area (C) increased by day 2. Although the follicles had grown, follicular diameter and area were comparable between the treatment and control groups on days −1 and 0. On day 1, follicular diameters were greater in the treatment group than in the control group, although the difference was not statistically significant. Mean±SEM (n=4). *p<0.05; **p<0.01.
The number of follicles increased in response to the superovulation treatment; however, there was no change in the follicular area immediately before ovulation (Figure 2). Before PGF2α administration on day −6, the follicular area in the treatment group was 0.23±0.01 cm2, while in the control group, it was 0.24±0.01 cm2, showing no significant difference (p=0.706). In response to serial FSH administration, the follicular area on day −2 in the treatment group was 0.81±0.03 cm2, while in the control group, it was 0.63±0.04 cm2, showing a significant increase (p =0.013). However, following hCG administration on day −1, the follicular area in the treatment group was 0.82±0.02 cm2, while in the control group was 0.77±0.05 cm2, showing no statistical difference (p=0.823). On estrus day (day 0), the follicular area was 0.77±0.02 cm2 in the treatment group and 0.65±0.06 cm2 in the control group (p=0.264). By the next day after estrus (day 1), the follicular area slightly increased to 0.90±0.05 cm2 and 0.74±0.08 cm2 in the treatment and control groups (p=0.955), respectively. However, no significant differences were observed in the estrus and post-estrus day measurements.
Similar to the changes observed in the follicular area, no significant differences were observed in the final follicular diameter after superovulation treatment (Figure 2). Serial administration of FSH led to a significant increase in follicular diameter on day −2, reaching 3.4±0.1 mm in the treatment group compared to 2.7±0.1 mm in the control group (p=0.004). However, on day 0, the day of estrus, the follicular diameters were 4.0±0.07 mm in the treatment group and 3.0±0.10 mm in the control group, showing no statistically significant difference (p= 0.324). This indicates that the final follicular diameter immediately before ovulation did not differ between the two groups. The results for follicular area and diameter suggest that follicular growth in the treatment group was within the normal physiological range, indicating that the administered FSH dose was appropriate and that this superovulation protocol effectively promoted normal follicular development.
Experiment 2: Evaluation of retrieved zygotes. As the ovarian response to the superovulation protocol became evident, fertilized embryos were collected via oviductal flushing after ovulation was confirmed by ultrasonography, and their quality was assessed as “experiment 2” to evaluate the efficacy of the protocol. Similar to the outcomes observed in other animals following superovulation treatment, individual variations in response to treatment were apparent. In this experiment, all collected eggs were at the single-cell stage (Table I, Figure 3).
Quality and quantity of the zygotes retrieved by oviduct flushing.
Development of fertilized embryos collected by oviduct flushing after superovulation treatment. Fertilized embryos collected via oviduct flushing exhibited a clear zona pellucida and a visible second polar body. Cleavage progressed smoothly, and the embryos developed to the blastocyst stage at a higher rate than in vitro-fertilized embryos derived from slaughterhouse-derived oocytes. The diameters of the collected zygotes ranged from 100 to 110 μm, whereas those of the resulting blastocysts ranged from 150 to 200 μm, both of which were within the normal range. (A) Day 0 zygotes, (B) Day 3 zygotes, and (C) Day 6 blastocysts.
During laparotomy, the number of CH on the surfaces of both ovaries was visually counted, yielding a mean of 50.3±10.4 (mean±SEM). Superovulation protocol resulted in an enhanced ovulatory response, compared to the normal ovulation rate previously reported in microminipigs (5.47±1.74 per animal) (23). The mean number of zygotes collected per animal via oviduct flushing was 14.7±3.2, corresponding to a recovery rate of 29.5±3.4% based on the number of CH (Table I). Considering that the average litter size in microminipigs is five, superovulation treatment was presumed to have increased the number of ovulations by approximately three times.
The collected zygotes were cultured in PZM-5 medium from day 1 to 3, and subsequently in PBM from day 4 to 6. Embryo development was evaluated based on the cleavage and blastocyst formation rates (Table I). The average number of embryos per animal was 14.7±3.2 (range=9-20), with a cleavage rate of 62.8±19.4% and blastocyst formation rate of 59.4±21.9%.
Discussion
In this study, we established a foundational superovulation protocol for collecting oocytes and fertilized embryos from microminipigs for reproductive engineering. The validity of this protocol was supported by the clear effect of FSH, which was particularly evident in the increased number of follicles. Moreover, the amount of FSH administered did not lead to a larger follicular size, suggesting that the dose was appropriate. Consistent with this, we successfully obtained more fertilized embryos than those observed in microminipigs with a normal estrous cycle (24, 25).
The effects of exogenous FSH appeared early during follicular development. On day −2, the follicular area and diameter (i.e., follicle size) in the treatment group were significantly larger than those in the control group. This increase in follicle size is likely due to FSH administration during the early phase of the estrous cycle when endogenous FSH secretion remains low under normal conditions. Supporting this, no significant difference in follicle size was observed between the treatment and control groups after day −1, when endogenous FSH levels are thought to rise naturally, even in a normal cycle (8, 26). Therefore, the difference in follicle size observed on day −2 was considered to reflect the expected response to exogenous FSH stimulation.
Although the effect of FSH was within the normal range in most follicles with normal follicular area and diameter, it was excessive in certain follicles observed on day 1 after ovulation. The larger follicle size observed in the treatment group on day one was attributed to the residual effects of exogenous FSH (20). The presence of cyst-like follicles is a foreseeable phenomenon following FSH administration; therefore, future modifications to the protocol are necessary to improve outcomes.
In this study, we established a foundational protocol for inducing superovulation in microminipigs. However, two key points that require improvement have become evident for the efficient collection of high-quality oocytes or embryos in microminipigs. First, the stepwise administration of FSH is labor intensive, and second, surgical laparotomy is required for retrieving oocytes or embryos.
Stepwise administration of FSH for superovulation has also been performed in cattle (21). However, protocols using pregnant mare serum gonadotropin (PMSG) are more commonly adopted in these animals because its long half-life allows for effective induction with a single injection, making it less labor-intensive (27, 28). The primary objective is to collect high-quality oocytes and embryos, which should never be overlooked. Although microminipigs weighing 20-30 kg are considered small pigs, they are relatively large for experimental use. Therefore, the development of a protocol using PMSG may offer a practical solution to reduce handling and labor.
In the present study, the number of follicles measured by ultrasound in experiment 1 was higher than the observed count of CH in experiment 2. It is assumed that ultrasound imaging measures the number of ovarian follicles present at a given time; however, not every follicle proceeds to ovulation. Consequently, the follicle count obtained via ultrasound tends to exceed the number of resulting CH. This difference appears to be due to follicular atresia, a natural degenerative process that eliminates follicles before ovulation (29, 30). Additionally, variability in follicle and CH counts between experiments may reflect individual differences between the microminipigs used in experiments 1 and 2.
In animal-based research, it is essential to minimize invasiveness as much as possible. In this study, the establishment of a superovulation protocol that allows for the collection of an increased number of zygotes from a single pig can be considered a step closer to achieving the principles of the 3Rs (replacement, reduction, and refinement) in experimental animals (31, 32). However, laparotomy is required to collect pig zygotes. Thus, future research should focus on refining the superovulation protocol and developing non-invasive Ovum Pick-Up (OPU) technique in microminipigs to enable the acquisition of high-quality embryos, with attention to animal welfare.
Translational research using large animals is essential for rapidly advancing the practical application of life sciences (33, 34). In such research, it is not sufficient to simply use animal subjects; rather, it is important to develop and utilize models that incorporate biotechnological approaches comparable to those used in basic research. In conclusion, the development of a method to efficiently collect high-quality oocytes and fertilized embryos will make the application of basic research findings more feasible in the future. This study provides valuable insights into the development of translational research models.
Acknowledgements
This work was supported by JSPS KAKENHI (Grant Number JP23K23784).
Footnotes
Authors’ Contributions
P.W. wrote-original draft, performed the experiments and data curation. K.H., I.S. and T.K. planned and performed the experiments and data analysis. H. K. reviewed and edited, validated, and supervised. M. T. reviewed and edited, validated, supervised, and conceptualized.
Conflicts of Interest
The Authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received September 19, 2025.
- Revision received October 10, 2025.
- Accepted October 20, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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