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,

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. Al....

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