Isoflurane-Assisted Electroacupuncture in a Rat Model of Oligoasthenozoospermia

Summary

This protocol details the establishment of an adenine-induced oligoasthenozoospermia model in rats and the subsequent delivery of electroacupuncture at CV3, CV4, ST36, and SP6 under isoflurane anesthesia. Outcomes were verified using computer-assisted sperm analysis for count and motility, alongside testicular histology with hematoxylin and eosin staining.

Abstract

Oligoasthenozoospermia is one of the most common forms of male reproductive dysfunction. In rodent studies, delivering electroacupuncture (EA) consistently across repeated sessions poses a technical challenge, particularly when combining abdominal midline and hind-limb acupoints, where stable electrode contact is critical. Zhongji (CV3), Guanyuan (CV4), Zusanli (ST36), and Sanyinjiao (SP6) were selected because these points are frequently used in acupuncture protocols for infertility and semen abnormalities, and the abdominal midline plus hind-limb configuration suits standardized electrode pairing. This report describes a practical protocol for establishing an adenine-induced rat model of oligoasthenozoospermia and for administering isoflurane-assisted EA with paired stimulation of CV3–CV4 and ST36–SP6. Male Sprague-Dawley rats received adenine by daily gavage for 28 days to induce reproductive impairment. Subsequently, EA was administered daily for 28 days under inhalational anesthesia, using 3% isoflurane for induction and 1.5% for mask maintenance, to enable stable needle placement and electrode pairing (CV3–CV4 and ST36–SP6) with defined stimulation parameters (2 Hz, 1.5 mA, 30 min). Treatment effects were evaluated via computer-assisted semen analysis for sperm count and motility, supplemented by testicular hematoxylin and eosin (HE) histology. Representative results indicate that adenine exposure reduced sperm count and motility and disrupted seminiferous tubule architecture, while EA was associated with partial recovery in both semen parameters and testicular morphology. This protocol offers a ready reference for studies requiring consistent EA delivery and semen/histological verification in a rat model of oligoasthenozoospermia.

Introduction

Male factors contribute substantially to infertility, accounting for approximately half of all infertile couples, with abnormalities in sperm count and motility being among the most common clinical findings^1,2. Oligoasthenozoospermia, characterized by reduced sperm concentration and impaired motility, is one of the most common forms of male reproductive dysfunction^3,4. Despite the availability of assisted reproductive techniques and empirical medical therapies, effective strategies to improve semen quality remain limited, highlighting the need for furt....

Protocol

All animal procedures complied with institutional guidelines for the care and use of laboratory animals and were approved by the Ethics Committee of Ningxia Medical University (approval no. IACUC-NYLAC-2021-130). Male Sprague-Dawley rats, aged 8 weeks and weighing 180–220 g, were obtained from the Experimental Animal Center of Ningxia Medical University (license no. SYXK (Ning) 2020-0001). The animals were housed under standard laboratory conditions at 22–24 °C and 52%–56% relative humidity with a 12 h light/12 h dark cycle, and they had free access to food and water.

1. Animal preparation, group assignment, and....

Results

Sperm analysis (Figure 3)
Epididymal sperm suspensions were analyzed using a computer-assisted semen analysis system to quantify sperm count and motility. In this representative dataset, adenine administration markedly reduced both parameters relative to the control group, confirming successful model establishment. EA intervention subsequently increased sperm count and motility compared to the model group (Figure 3). One-way ANOVA indicated significant group effects for .......

Discussion

Given the multifactorial etiology of oligoasthenozoospermia and the limited efficacy of available pharmacologic treatments, acupuncture and EA have gained attention as repeatable, nonpharmacological interventions¹⁹. The existing clinical and preclinical literature suggests several plausible mechanisms by which acupuncture or EA may influence male reproductive function, including the modulation of neuroendocrine regulation, improvement of reproductive organ microcirculation, and attenuation of oxid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenine	Beijing Solarbio Science & Technology Co.Ltd.	A8330
CASA computer-assisted semen analysis system	Nanning Songjing Tianlun Biotechnology Co.,Ltd.	VICOS-SPERM
Centrifuge	Sartorius	A-14C
Differentiation fluid	Shanghai Biyuntian Biotechnology Co.,Ltd	C0163M
Disposable sterile acupuncture needles	Hanyi brand		0.25 mm ´ 13 mm
Drying machine	Leica	HI1220
Electrical stimulator	Changzhou Yingdi Electronic Medical Device Co., Ltd	KWD-808I
Electronic balance	Hangzhou Youheng Weighing Equipment Co.,Ltd	HLD-6002
Eosin Y Stain Solution	Lanke Biotech	BS248
Hematoxylin staining solution	Zhuhai Beso Biotechnology Co. LTD	BA-4041
Isoflurane	RWD Life Science Co., Ltd.	R510-22-10
Microtome	Leica	RM2255
Neutral balsam	Beijing Zhongshan Jinqiao Biotechnology Co.,Ltd	Zli-9555
Paraffin embedding machine	Leica	HistoCore Arcadia H
SD rats	Animal Center of Ningxia Medical University		8-week-old,Male,180-220 g
Slide scanner	Leica	Aperio LV1
Small Animal Anesthesia Machine	RWD Life Science Co., Ltd.	R500
Vortex oscillator	Haimen Qilin Bell Instrument Manufacturing Co.,Ltd	QL-902
Warming pad	Dongguan Hongda Electronics Co., Ltd.	45W-80X28CM