A Simple Microaspiration Technique for Isolating Somatic Cells from Cryopreserved Equine Semen as Nuclear Donors for Cloning

Summary

This protocol presents a simple microaspiration technique to isolate viable somatic cells (SCs) from cryopreserved equine semen. Unlike traditional approaches, this technique avoids long in vitro cultures and overcomes sperm contamination, enabling immediate use in somatic cell cloning.

Abstract

Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures from these isolated cells. Furthermore, the samples that were obtained contained a high load of spermatozoa, which interferes with the establishment and maintenance of in vitro cultures. To date, primary cultures have only been successfully established from fresh semen samples, while attempts using cryopreserved semen have consistently failed. This limitation significantly restricts the potential to generate clones from cryopreserved semen samples. The present study proposes a simple microaspiration-based methodology for isolating somatic cells from cryopreserved equine semen. This approach allows for the rapid retrieval of a sufficient number of cells for immediate use in SCNT cloning procedures.

Introduction

Somatic cell nuclear transfer cloning is an invaluable tool for the rescue and multiplication of individuals of zootechnical and scientific interest. Numerous cell lines have been used as nuclear donors, including cells derived from skin, muscle, granulosa, lymphocytes, and urine cells^1,2,3,4. Interestingly, semen has emerged as a novel alternative for collecting somatic cells (SCs) to be used as nuclear donors for cloning^5,6. This finding is particularly important for the rescue of ....

Protocol

Cryopreserved semen samples, preserved for over 19 years in liquid nitrogen, were obtained from commercial distributors authorized by the relevant veterinary authorities in Mexico, in compliance with the Mexican Official Standard NOM-027-ZOO-1995, which governs the zoosanitary process of semen from domestic animals. All semen samples, whether used or unsuitable for analysis, were inactivated and disposed of following institutional biosafety protocols and applicable regulations, including NOM-087-ECOL-SSA1-2002 for the management of biological-infectious waste in Mexico. Residual hydrofluoric acid (HF) was safely neutralized under a fume hood by slowly transferring it ....

Results

Table 1 shows that the microaspiration technique efficiently recovers somatic cells from cryopreserved equine semen, with capture rates ranging from 317.7 ± 9.60 to 424.7 ± 33.65 cells/hour across three stallions. Stallion #03 had the highest yield, demonstrating the effectiveness of the method even with long-term stored samples. The data indicate that the technique consistently and effectively isolates somatic cells from semen, and the mean capture rates and standard dev.......

Discussion

Micromanipulation of cells and gametes has made it possible to simplify complex assisted reproductive techniques. Examples of these include somatic cloning by Hand Made Cloning²¹, assisted hatching²², intracytoplasmic sperm injection (ICSI)²³, and pronuclear microinjection in zygotes²⁴, among others.

The development of effective methods for isolating somatic cells from semen is an invaluable tool fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 beta Mercaptoethanol	SIGMA	M7522	Somatic cells culture
1x DPBS	GIBCO	REF-21600010	Somatic cells manipulation
Absolute ethanol	HYCEL	EL CRISOL 50181-1L73	For fabricating pipettes
Antibiotic-antimicotic 100x	INVITRO SA. México		Somatic cells culture
Borosilicate capillaries (1.0 mm O.D. × 0.78 mm I.D. × 100 mm Length)	HARVARD APPARATUS, USA	EC1-30-0035	For fabricating pipettes
BSA	SIGMA	A3311	Somatic cells culture
DMEM/F12 medium	GIBCO	REF-12500-062	Somatic cells culture
Equine cryopreserved semen sample			The cryopreserved semen samples were acquired from a commercial distributor and had been preserved for more than 19 years in liquid nitrogen. All frozen equine semen samples were obtained from commercial distributors authorized by the relevant veterinary authorities in Mexico, in accordance with the Mexican Official Standard NOM-027-ZOO-1995, Zoosanitary Process of Semen from Domestic Animals.
Eyepiece reticle		Tool for fabricating pipettes	Required equipment
Fetal Bovine Serum	GIBCO	REF-26140-079	Somatic cells culture
Hydrofluric acid	SIGMA-ALDRICH	339261	For fabricating pipettes
Invert optical Microscope	Eclipse, TE 200	NIKON	Somatic cells micromanipulation
Laboratory  dry oven		Tool for fabricating pipettes	Required equipment
Microforge		Tool for fabricating pipettes	Required equipment
Micromanipulator set for Inverted Microscope		Micromanipulation of cells	Required equipment
Micropipette puller	P-97, SUTTER INSTRUMENT	Tool for fabricating pipettes	Use following settings—Heat: 720; Pull: 30; Velocity: 50; Time: 220; Pressure
Mineral oil	SIGMA	M8410	Somatic cells manipulation
Nunclon cell culture dishes, diam. × H 35 mm × 10 mm	NUNCLON	Nunc 153066
PVA	SIGMA	P8136	Somatic cells culture
PVP	SIGMA	P5288	Somatic cells culture
Trypan blue solution 0.4%	GIBCO	REF- 15250061	Cell dye