Massive Ovulation in Mammals: Techniques for Analyzing Natural Spontaneous Polyovulation and Induced Polyovulation in Lagostomus maximus

Summary

This protocol describes methods for analyzing natural and induced ovulation in Lagostomus maximus, including oocyte recovery after spontaneous, hormonal-induced, or seminal plasma-induced ovulation. It provides a comprehensive framework for studying ovarian responsiveness and reproductive physiology in a highly polyovulatory mammalian model.

Abstract

The South American plains vizcacha, Lagostomus maximus, is recognized as the mammalian species with the highest ovulation rate, which can reach as many as 300 ova. This remarkable polyovulation phenomenon can be examined through several methodological approaches that reflect its distinctive reproductive physiology. Natural ovulation is assessed by oocyte release during the breeding season, which provides insights into the species' spontaneous ovulatory mechanisms, with oocytes recovered by flushing the oviduct and uterine horns. In addition, experimental induction of ovulation using exogenous hormones, such as gonadotropins, allows controlled evaluation of ovarian responsiveness and offers a valuable model for understanding endocrine regulation in hystricognath rodents. A third approach involves the use of autologous seminal plasma as a physiological trigger of ovulation, highlighting the coexistence of induced and spontaneous mechanisms in this species. Together, these techniques not only elucidate the dynamics of follicular recruitment and oocyte release but also establish L. maximus as a comparative model for reproductive biology, with potential translational implications for understanding ovulation control in mammals.

Introduction

The South American plains vizcacha (Lagostomus maximus) is a hystricognath rodent distinguished by its exceptional reproductive physiology, characterized by an extraordinary ovulation rate that can exceed 300 oocytes per cycle^1,2. This distinctive feature has positioned L. maximus as a natural model for studying ovarian function and ovulatory control in mammals^{3,4,5,6,7,8,9,10,11}.

Unlike typical spontaneous ovulators, L. maximus exhibits both spontaneous and induced ovulatory mechanisms². This duality, combined with massive follicular growth and asynchronous ovulation, challenges conventional approaches based solely on endocrine or histological endpoints^4,12,13,14. Consequently, the use of direct, quantitative techniques is essential to accurately determine the timing and magnitude of oocyte release.

Adult female vizcachas were obtained from a natural resident population maintained at the Estación de Cría de Animales Silvestres (ECAS), Parque Pereyra Iraola, Berazategui, Buenos Aires Province, Argentina (34°49′57″ S, 58°06′12″ W). We acknowledge that sovereign rights over natural resources are the exclusive property of the Province of Buenos Aires. The number of captured animals was authorized by the Ministry of Agriculture of Buenos Aires Province. Females were captured using live traps placed at burrow entrances at different time points throughout the year.

Capture periods were selected according to the species natural reproductive cycle established by Llanos & Crespo¹⁵, and refined based on our fieldwork experience^7,8,10,14. The species presents a main reproductive season that extends from late summer (March) until the beginning of springtime in late September^16,17,18, and a secondary breeding period that occurs in October, especially in females that have lost their offspring¹⁵. Accordingly, the following working groups were determined: a) Group I (N= 27): late February-early April (non-pregnant, onset of main reproductive season); b) Group II (N= 26): mid-September-late October (non-pregnant, post-lactation estrus); Group III (N= 35): December-January (non-pregnant, hormone-induced ovulation); Group IV (N= 7): December-January (non-pregnant, seminal plasma-induced ovulation).

This study presents and validates several methodological approaches for analyzing ovulation in L. maximus: (1) assessment of natural ovulation through direct oocyte recovery; (2) induction of ovulation using exogenous gonadotropins; and (3) induction of ovulation with autologous seminal plasma. Each technique provides complementary insights into follicular dynamics, oocyte maturation, and the regulatory mechanisms governing ovulation.

Flushing of the oviducts and uterine horns enables direct recovery of oocytes and accurate quantification of ovulation, allowing correlation with follicular development and oocyte morphology^19,20,21. Hormonal stimulation with exogenous gonadotropins, adapted from protocols used in laboratory rodents²², allows controlled evaluation of ovarian responsiveness and the timing of oocyte release. Meanwhile, the use of autologous seminal plasma as a physiological trigger of ovulation -previously described in induced ovulators such as rabbits and camelids^23,24,25-offers an innovative means of exploring the endocrine and paracrine factors involved in ovulation.

By integrating these complementary methodologies, the present work provides a reproducible framework for investigating ovulatory mechanisms in L. maximus. The combination of natural and induced models allows differentiation between spontaneous follicular recruitment, ovulatory efficiency, and seminal plasma-mediated responses. Moreover, these approaches reveal phenomena such as spontaneous parthenogenetic oocyte activation and the coexistence of "defective" and "euovulatory" oocytes, contributing to the understanding of follicular selection and oocyte competence².

Beyond its species-specific implications, the vizcacha model offers broader relevance for comparative reproductive biology. Its mixed ovulatory strategy bridges characteristics of induced and spontaneous ovulators, providing valuable parallels to reproductive processes in other mammals, including domestic species and humans^7,14,26,27.

The methodological protocols detailed herein are designed to ensure reproducibility and to facilitate their adaptation to diverse experimental contexts involving follicular dynamics, ovulatory regulation, and hormonal manipulation.

Protocol

The experimental protocols described herein were reviewed and approved by the Institutional Committee on the Use and Care of Experimental Animals (CICUAE, Universidad Maimónides, protocol number #6184). Trapping, handling, and euthanasia were performed by trained technical staff in compliance with all local, state, and federal regulations governing the care and use of laboratory animals. Animal husbandry followed the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals²⁸ and the guidelines of the American Society of Mammalogists (ASM) for the use of wild mammals in research²⁹. All experiments complied with the ARRIVE 2.0 guidelines³⁰. Every effort was made to minimize the number of animals used. The South American plains vizcacha (Lagostomus maximus) is not considered an endangered species³¹.

1. Hormonal induction of ovulation

​NOTE: This section presents hormonal induction of ovulation in non-pregnant females of captured vizcachas. Three different protocol options are discussed.

1. Protocol A:
   1. Administer 200 IU of equine chorionic gonadotropin (eCG) intramuscularly at 0 h and 24 h, followed by 500 IU of human chorionic gonadotropin (hCG) at 48 h.
   2. Rest females until euthanasia.
   3. Euthanize females 60-132 h post-hCG administration (see section 3).
2. Protocol B:
   1. Administer 200 IU of eCG intramuscularly at 0 h and 24 h, followed by 1,000 IU of hCG at 72 h.
   2. Rest females until euthanasia.
   3. Euthanize females 60-132 h post-hCG administration (see section 3).
3. Protocol C:
   1. Administer 250 IU of eCG intramuscularly at 0 h, 24 h, and 48 h, followed by 2 mg of porcine luteinizing hormone (pLH) at 72 h.
   2. Rest females until euthanasia.
   3. Euthanize females 60-132 h post-pLH administration (see section 3 and Table 1).
      NOTE: The animals in each group were euthanized at different time points (ranging from 60 h to 132 h post-hCG administration). This temporal distribution was essential to confirm the extended nature of the ovulation period in this species. Specifically, for experiments aiming for the exclusive collection of mature oocytes, euthanasia was consistently performed at 120 h post-hCG.

2. Induction of ovulation by seminal plasma

NOTE: Test the capacity of seminal plasma to induce ovulation by intramuscular administration in hormonally primed females.

1. Semen collection and preparation
   1. Collect semen from reproductively active males by electroejaculation, as described by Giacchino³².
   2. Centrifuge semen at 200 × g for 10 min at room temperature to pellet sperm cells and cellular debris.
   3. Supplement supernatants (seminal plasma) with penicillin and streptomycin and store at -20 °C until use.
2. Induction of ovulation in non-pregnant females by seminal plasma
   1. Administer 200 IU of eCG intramuscularly at 0 h and 24 h for each female.
   2. Inject seminal plasma intramuscularly 48 h after the final eCG dose.
   3. Rest females until euthanasia.
   4. Euthanize animals 72 h after seminal plasma injection (see section 3).
      NOTE: Include control females injected with saline solution instead of seminal plasma.

3. Anesthesia, euthanasia, and reproductive tract collection

NOTE: Use anesthesia and humane euthanasia methods to obtain intact reproductive tracts for oocyte and histological analyses.

1. Induce euthanasia via a single, calculated intramuscular injection of 13.5 mg/kg ketamine chlorhydrate and 0.6 mg/kg xylazine chlorhydrate administered according to the manufacturer's recommendations and established institutional protocols.
2. Confirm deep anesthesia by the absence of response to pain (pedal reflexes).
3. Place the animal in the supine position and sterilize the abdomen.
4. Surgically open the abdominal cavity to fully expose all abdominal organs.
5. Perform a general inspection to determine the overall condition of the animal and confirm natural or hormone-induced ovulation by the presence of ovulatory stigmata.
6. Obtain a blood sample by direct puncture of the superior vena cava for serological testing.
7. Perform euthanasia on the animal by administering 0.5 mL/kg of sodium pentobarbital intracardially.
8. Carefully dissect and remove the ovaries, oviducts, and uterine horns, taking care not to damage their structures.
9. Keep tissues in ice-cold phosphate-buffered saline (PBS) until processing (≤15 min).

4. Oocyte recovery

NOTE: Recover oocytes from the oviducts and uterine horns by gentle flushing to ensure complete collection of released gametes while minimizing tissue damage.

1. Prepare the flushing apparatus by attaching a blunted 26-G needle to a 1 mL syringe filled with 0.9% NaCl supplemented with 4 mg/mL BSA, 100 µg/mL penicillin G, and 100 IU/mL streptomycin.
2. Gently insert the needle into the infundibulum of each oviduct and slowly inject the flushing solution.
3. Collect the effluent from the distal oviduct and uterine horn into sterile Petri dishes.
4. Repeat flushing until the recovered fluid appears clear and free of visible oocytes or cellular debris.
5. Transfer oocytes to 100 µL droplets of M2 medium containing 4 mg/mL BSA and antibiotics under mineral oil.
6. Confirm successful recovery by inspecting droplets under a stereomicroscope; expect to observe free oocytes lacking cumulus cells dispersed within the fluid.
7. Continue flushing until the effluent appears clear and no additional oocytes are observed under the stereomicroscope (typically 3-5 flushes per oviduct).
   NOTE: Recovered oocytes should appear spherical and translucent under low magnification.
8. Classify oocytes according to morphological integrity as described in section 6.

5. Oocyte morphological assessment

NOTE: Evaluate the quality of recovered oocytes based on cytoplasmic appearance, zona pellucida uniformity, and nuclear maturation stage.

1. Identify oocytes as normal if they display a spherical shape, uniform zona pellucida, homogeneous translucent cytoplasm, and a visible germinal vesicle or first polar body.
2. Identify oocytes as abnormal if the cytoplasm appears dark, granular, or fragmented, or if the zona pellucida is irregular.
3. Record the nuclear maturation stage as germinal vesicle (GV), germinal vesicle breakdown (GVBD), or metaphase II (MII).

6. Evaluation of oocyte nuclear maturation and meiotic spindle integrity

NOTE: Assess nuclear maturation and spindle morphology by immunofluorescence staining of α-tubulin and chromatin counterstaining.

1. Fix oocytes in 2% paraformaldehyde (PFA) for 20 min.
2. Permeabilize in 0.2% Triton X-100 in PBS for 40 min.
3. Post-fix in 2% PFA for 20 min and wash three times in PBS (30 min each).
4. Block with 2% goat serum, 2% BSA, 2% milk powder, 0.1 M glycine, and 0.01% Triton X-100 in PBS for ≥ 1 h.
5. Incubate oocytes overnight at 4 °C with mouse anti-α-tubulin antibody (1:200).
6. Wash in PBS for 30 min and incubate for 1 h in darkness with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1/1000).
7. Counterstain with 10 µg/mL Hoechst 33324 for 10 min and wash in PBS containing 2% BSA.
8. Mount oocytes in antifade medium and image using a confocal microscope.
9. Capture and process images using EZ-C1 software (EZ-C1 version 3.90); adjust contrast and brightness in image-processing software as required.
   NOTE: Successful staining is indicated by a clear green fluorescence pattern of the meiotic spindle (FITC) and blue nuclear counterstaining with Hoechst 33324.

7. Statistical analysis

NOTE: Analyze quantitative data to compare ovulatory responses and oocyte quality among experimental groups.

1. Express data as mean ± SD.
2. Use Student's t-test for two-group comparisons.
3. Apply one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons.
4. Consider p < 0.05 statistically significant.

Results

Natural ovulation

Twelve out of 27 females (group I) captured from late February to mid-April, coinciding with the primary mating season, were observed to be ovulating, whereas during the secondary mating season (September/October) (group II), 10 out of 26 captured females exhibited ovulation. A comparable success rate of approximately 40% of ovulating females was observed during both capture periods. No swelling of the ampullary region of the oviduct was noted in any instance...

Discussion

The protocol presented here for studying ovulation in the plains vizcacha provides a reproducible framework for assessing both natural and experimentally induced ovulatory events. A major advantage of this protocol lies in its capacity to directly quantify and characterize the extraordinarily high number of oocytes released by the plains vizcacha, a feature unmatched in other mammalian models. Unlike indirect methods based on hormonal profiles or histological sections, oviductal and uterine flushing provides immediate ac...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adobe Photoshop	Adobe Systems Inc., USA	N/A	Image processing
Bovine Serum Albumin (BSA)	Sigma-Aldrich, USA	A9647	Used in flushing medium and blocking buffer
Centrifuge (for semen processing)	Eppendorf, Germany	5417R	Seminal plasma preparation
Confocal Microscope (C1 Eclipse E800)	Nikon Ltd., Japan	N/A	Oocyte imaging
Electro-ejaculator	Designed in the laboratory; adapted from domestic mammals for use in caviomorph rodents	N/A	Semen collection
Euthanyl (sodium pentobarbital, sodium diphenylhydantoin)	Brouwer S.A., Argentina	N/A	Euthanasia
FITC-conjugated goat anti-mouse IgG (AP124F)	Chemicon, USA	AP124F	Secondary antibody
Hematoxylin-Eosin staining kit	Sigma-Aldrich, USA	Various	Histology
Hoechst 33324	Thermo Fisher Scientific, USA	H3570	Nuclear staining
Ketamine chlorhydrate	Holliday Scott S.A., Argentina	N/A	Anesthesia
Laminar flow hood	Thermo Scientific, USA	N/A	Aseptic handling
Light microscope	Olympus, Japan	BX51	Histology
Lutropin-V (pLH)	Bioniche Animal Health Care, Canada	N/A	Hormonal induction
M2 medium	Sigma-Aldrich, USA	M7167	Oocyte recovery
Mineral oil	Sigma-Aldrich, USA	M8410	Oocyte culture
Novormon 5000 (eCG)	Syntex S.A., Argentina	N/A	Hormonal induction
Ovusyn (hCG)	Syntex S.A., Argentina	N/A	Hormonal induction
Paraformaldehyde (PFA, 2% and 4%)	Sigma-Aldrich, USA	158127	Fixation
Penicillin G	Sigma-Aldrich, USA	P3032	Antibiotics for media
Stereomicroscope	Leica Microsystems, Germany	M80	Oocyte identification
Streptomycin	Gibco, Thermo Fisher Scientific, USA	11860038	Antibiotics for media
Syringe with blunt-end needle (1 mL)	BD Biosciences, USA	305219	Flushing of oviducts/uterus
Triton X-100	Sigma-Aldrich, USA	T8787	Permeabilization
VectaShield mounting medium	Vector Laboratories, USA	H-1000	Mounting for fluorescence
Xylazine chlorhydrate	Richmond Laboratories, Argentina	N/A	Anesthesia
Xylene	Sigma-Aldrich, USA	534056	Dewaxing paraffin sections
α-tubulin monoclonal antibody (mouse anti-α-tubulin, sc-5286 B7)	Santa Cruz Biotechnology, USA	sc-5286 B7	For meiotic spindle immunostaining