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Method Article

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

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DOI:

10.3791/69404

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December 19th, 2025

 ,  ,  , 
1Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, 2Departamento de Producción Agrícola y Animal, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Xochimilco, División de Ciencias Biológicas y de la Salud, 3Integrated Cell Culture & Bioimaging Laboratory, Division of Science, Mathematics & Agribusiness, Mayville State University, 4Conservation, Genetics & Biotech, LLC, 5Laboratorio de Manejo de la Reproducción Animal, Departamento de Producción Agrícola y Animal, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Xochimilco

In This Article

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 cells1,2,3,4. Interestingly, semen has emerged as a novel alternative for collecting somatic cells (SCs) to be used as nuclear donors for cloning5,6. This finding is particularly important for the rescue of individuals for whom only semen has been preserved. However, the establishment of primary cultures from SCs isolated from frozen semen has been unsuccessful and, to date, remains the main obstacle for obtaining cloned animals from this source7,8,9. These cells likely originate from glandular epithelia and the lining of tubules, from seminal secretions, and-importantly-from semen contamination with urine, the latter being a well-known source of somatic cells with high proliferative and undifferentiated potential3.

Various methods for isolating SCs from semen have been implemented, including centrifugation5, osmotic gradients7,8,10, and flow cytometry11. However, somatic cells, once isolated by these methods, still require optimized conditions for attachment, growth, and proliferation8. The specific requirements of these cells make in vitro culture more complex, as prolonged incubation periods are needed for the establishment of primary cell cultures9. Moreover, the high concentration of spermatozoa present in the samples interferes with both the initiation and maintenance of these cultures5.

In contrast to previously described approaches, this microaspiration technique offers several clear advantages. It permits the selection of SCs based on size and morphology12 and allows the recovery of nuclei from both viable and nonviable cells, a strategy successfully demonstrated with frozen tissue without cryoprotectants13 and freeze-dried reproductive cells14,15,16. Importantly, SCs can be used immediately after semen thawing, thus reducing the risk of ploidy errors typically associated with extended in vitro culture of primary somatic cells used for animal cloning17,18. Specifically, the technique focuses on cryopreserved equine semen, allowing the collection of a sufficient number of SCs to be used immediately for SCNT12.

The fabrication of micropipettes and the development of microaspiration techniques have significantly progressed in recent decades. The use of these tools enables precise manipulation of gametes, embryos, and their cytostructures, creating new possibilities in the field of reproductive biotechnology (https://www.jove.com). Micromanipulation techniques enable the evaluation of cellular mechanical properties and provide valuable insights into their physiological condition19,20, yet this field is still relatively unexplored.

To date, no research has detailed an effective technique for isolating SCs from semen using any variant of micromanipulation procedures. Given the limitations of conventional isolation methods, this study proposes a practical microaspiration-based protocol to isolate SCs from cryopreserved equine semen, enabling their immediate use in somatic cloning procedures following collection.

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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 into a plastic container and gradually adding small portions of CaCO3 or Ca(OH)2 until effervescence ceased, forming insoluble CaF2. The pH of the neutralized suspension was confirmed to be between 6 and 8 before it was managed as inorganic chemical waste according to local regulations. The reagents and the equipment used are listed in the Table of Materials.

1. Micropipette needle fabrication for somatic cell isolation

  1. Select borosilicate glass capillaries (1.0 mm O.D. × 0.78 mm I.D. × 100 mm).
  2. Use a micropipette puller to produce fine-tipped capillaries. Adjust the cutting point to vary the tip diameter.
  3. Insert the capillary into the puller and lock it firmly.
  4. Remove the micropipette carefully after pulling and place it horizontally on the microforge.
  5. Align the heater and micropipette within the microscope's field of view.
  6. Locate the cutting point using the eyepiece reticle (Figure 1A,B).
  7. Bring the micropipette tip into contact with the glass bead (on the heater filament).
  8. Gradually increase the heat until the tip melts into the bead.
    1. Turn off the heater.
    2. Allow the tip to adhere to the bead and separate.
      NOTE: It is not necessary to bend the micropipette; it can be used in its straight form. However, if preferred by the operator, an angle may be formed (Figure 1C).
  9. Do not smooth the tip, as this step is unnecessary for the procedure.
  10. Clean the pipette tips by immersing each tip for ~10 s sequentially in:
    1. 20% hydrofluoric acid (HF).
      ​CAUTION: Hydrofluoric acid is highly toxic and corrosive. Use the personal protective equipment indicated by the supplier. Emphasize the use of eye/face protection, gloves, and a laboratory coat, and always work in a fume Hood.
    2. Sterile double-distilled water.
    3. Absolute ethanol.
      NOTE: To facilitate pipette tip washing, prepare the above-mentioned solutions in 15-mL sterile Corning tubes.
  11. Dry micropipettes in an oven at 50 °C.
  12. Store micropipettes in a sterile container.

2. Microaspiration setup

  1. Check that the microaspiration system is in proper working condition, with the microaspiration joystick centered on the control panel.
  2. For systems using oil, ensure there are no air bubbles inside the system, as these may affect movement precision.
  3. Insert the somatic cell aspiration micropipette into the holding manipulator on the operator's dominant hand side.
    NOTE: Replace the micropipette with a new one if the tip becomes clogged during somatic cell collection.

3. Thawing and dilution of cryopreserved equine semen

  1. Remove straws from liquid nitrogen, expose to air for 10 s, and thaw at 37 °C for 30 s.
  2. First semen dilution: Take a sample from the thawed semen (250 µL) and dilute with 1.0 mL of DMEM/F12 medium supplemented with 10% fetal bovine serum, 0.1 mg/mL bovine serum albumin, 12.5 µM β-mercaptoethanol, and 1% antibiotic-antimycotic.
    1. Incubate the mixture at 37 °C for 30 min.
  3. Second semen dilution: Take 25 µL from the first dilution, dilute it in 975 µL of 1x DPBS supplemented with 0.1 mg/mL poly(vinyl alcohol) (PVA) or polyvinylpyrrolidone (PVP) deposit it into a 1.5 mL microcentrifuge tube, and mix gently.
    1. Maintain this mixture at 37 °C throughout the cell collection process.

4. Preparation of dishes for SCs collection

  1. Collect the SCs using two lids from 35 mm diameter culture dishes.
  2. Prepare the first lid as follows (lid #1, Figure 2A).
    1. Take 100 µL of the second semen dilution and deposit it in the center of the lid (see Figure 2A).
    2. Make a second microdrop (10 µL) of 1x DPBS supplemented with 0.1 mg/mL PVA or PVP on top of the first drop (see Figure 2A).
    3. Cover both microdrops completely with paraffin oil to prevent evaporation and to maintain temperature stability during SCs aspiration.
  3. Prepare the second lid to remove cellular debris and accompanying spermatozoa (lid #2, Figure 2B).
    1. Place three 30-50 µL drops of 1x DPBS supplemented with 0.1 mg/mL PVA or PVP in the center of the lid; do not cover them with mineral oil.
      ​NOTE: Prepare this culture dish only after all somatic cells have been selected to prevent changes in the physicochemical properties of the DPBS solution due to air exposure.
  4. Perform all microaspiration steps under an inverted microscope at room temperature. Avoid sudden temperature changes.

5. Microaspiration procedure for isolating SCs from cryopreserved equine semen

  1. Place the first lid (lid #1, Figure 2A) on the microscope stage, ready for collecting the SCs.
  2. Select a micropipette with the proper diameter to collect the SCs and connect it to the mechanical aspiration system.
  3. Carefully insert the micropipette into the microdrop and align it with the bottom of the dish lid. Once the pipette has aspirated culture medium, begin the individual capture of SCs.
  4. At the bottom of the dish, various cell morphologies should be visible (Figure 3).
  5. Position the micropipette tip adjacent to each SC and aspirate them under negative pressure. Aspirate all round and elongated cells regardless of their size. The aspiration volume varies between captures of SCs.
  6. After capturing 10 cells, deposit them into the microdrop located on top (10µL) (Figure 2A). Repeat this step until the required number of SCs for the cloning experiment is obtained.
  7. Hold all captured somatic cells within this microdrop throughout the collection process.
  8. Once the required number of SCs for cloning is obtained, perform a simple washing procedure using microdrops to remove accompanying spermatozoa, cellular debris, and microbial contaminants.
  9. Take the second dish lid and perform a sequential three-step washing of the SCs (lid #2, Figure 2B):
    1. Capture the somatic cells with a micropipette and carefully deposit them onto the surface of the first drop. Allow them to settle for 3-5 min (Figure 2B; Drop #1).
    2. Repeat step 5.9.1, and deposit them into the second drop (Figure 2B; Drop #2).
    3. Repeat step 5.9.1, and deposit the somatic cells into the third drop (Figure 2B; Drop #3).
  10. As a final step, deposit the isolated somatic cells into a final microdrop prepared for subsequent use in somatic cell nuclear transfer procedures (Figure 4D).
    NOTE: Aspirate SCs in the smallest possible medium volume during each recapture to minimize sperm, debris, and microbial contaminants.

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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...

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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...

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Disclosures

The authors declare that they have no conflicts of interest.

Acknowledgements

The authors thank the Laboratory of Reproduction Management, Department of Agricultural and Animal Production, Universidad Autónoma Metropolitana-Xochimilco (UAM-X), for their technical and logistical support.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2 beta MercaptoethanolSIGMAM7522Somatic cells culture
1x DPBSGIBCOREF-21600010Somatic cells manipulation
Absolute ethanolHYCELEL CRISOL 50181-1L73For fabricating pipettes
Antibiotic-antimicotic 100xINVITRO SA. MéxicoSomatic cells culture
Borosilicate capillaries (1.0 mm O.D. × 0.78 mm I.D. × 100 mm Length)HARVARD APPARATUS, USAEC1-30-0035For fabricating pipettes
BSASIGMAA3311Somatic cells culture
DMEM/F12 mediumGIBCOREF-12500-062Somatic cells culture
Equine cryopreserved semen sampleThe 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 reticleTool for fabricating pipettesRequired equipment
Fetal Bovine SerumGIBCOREF-26140-079Somatic cells culture
Hydrofluric acidSIGMA-ALDRICH339261For fabricating pipettes
Invert optical MicroscopeEclipse, TE 200NIKONSomatic cells micromanipulation
Laboratory  dry ovenTool for fabricating pipettesRequired equipment
MicroforgeTool for fabricating pipettesRequired equipment
Micromanipulator set for Inverted MicroscopeMicromanipulation of cellsRequired equipment
Micropipette pullerP-97, SUTTER INSTRUMENTTool for fabricating pipettesUse following settings—Heat: 720; Pull: 30; Velocity: 50; Time: 220; Pressure
Mineral oilSIGMAM8410Somatic cells manipulation
Nunclon cell culture dishes, diam. × H 35 mm × 10 mmNUNCLONNunc 153066
PVASIGMAP8136Somatic cells culture
PVPSIGMAP5288Somatic cells culture
Trypan blue solution 0.4%GIBCOREF- 15250061Cell dye

References

  1. Pandey, A., Gupta, S. C., Gupta, N. Comparative potential of cultured skin fibroblast, cumulus, and granulosa cell to produce somatic cell nuclear transfer (SCNT) preimplantation embryos in buffaloes (Bubalus bubalis) in relation to gene expressions. Cell Reprogram. 12 (3), 357-368 (2010).
  2. Galli, C., Duchi, R., Moor, R. M., Lazzari, G. Mammalian leukocytes contain all the genetic information necessary for the development of a new individual. Cloning. 1 (3), 161-170 (1999).
  3. Olsson, P. O., Yeonwoo, J., Park, K., Yoo, Y. M., Hwang, W. S. Live births from urine derived cells. PLoS One. 18 (1), e0278607(2023).
  4. Vignon, X., et al. Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. C R Acad Sci III. 321 (9), 735-745 (1998).
  5. Nel-Themaat, L., et al. Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope. Reprod Fertil Dev. 19 (4), 576-584 (2007).
  6. Kjelland, M. E., Liu, J., Hyatt, D., Kraemer, D. Collection, isolation, and culture of somatic cells from avian semen. Avian Biol Res. 6 (4), 255-260 (2013).
  7. Nel-Themaat, L., et al. Cloned embryos from semen. Part 1: in vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation. Cloning Stem Cells. 10 (1), 143-160 (2008).
  8. Liu, J., et al. Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen. Anim Reprod Sci. 115 (1-4), 49-57 (2009).
  9. Liu, J., et al. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen. Theriogenology. 74 (9), 1629-1635 (2010).
  10. Saini, M., et al. Isolation and culture of epithelial cells from stored buffalo semen and their use for the production of cloned embryos. Reprod Fertil Dev. 31 (10), 1581-1588 (2019).
  11. Brom-de-Luna, J. G., Canesin, H. S., Wright, G., Hinrichs, K. Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting. Anim Reprod Sci. 190, 10-17 (2018).
  12. Ramos, B., Rodríguez, A. A., Rosales, A. M., Montiel, A. J., Hernández, J. E. Identification of somatic cell populations in cryopreserved equine semen as a source of donor nuclei for somatic cloning: preliminary results. Reprod Fertil Dev. 37 (1), (2025).
  13. Moulavi, F., et al. Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes. Theriogenology. 90, 197-203 (2017).
  14. Wakayama, S., Ito, D., Ooga, M., Wakayama, T. Production of mouse offspring from zygotes fertilized with freeze-dried spermatids. Sci Rep. 12 (1), 18430(2022).
  15. Wakayama, S., Ito, D., Hayashi, E., Ishiuchi, T., Wakayama, T. Healthy cloned offspring derived from freeze-dried somatic cells. Nat Commun. 13 (1), 3666(2022).
  16. Loi, P., Fulka, J., Hildebrand, T., Ptak, G. Genome of non-living cells: Trash or recycle. Reproduction. 142 (4), 497-503 (2011).
  17. Wakayama, S., Wakayama, T., Wells, D. N. Encyclopedia of Life Sciences. , 1-10 (2023).
  18. Lanza, R., et al. Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science. 288, 665-669 (2000).
  19. González-Bermúdez, B., Guinea, G. V., Plaza, G. R. Advances in micropipette aspiration: applications in cell biomechanics, models, and extended studies. Biophys J. 116 (4), 587-594 (2019).
  20. Hochmuth, R. M. Micropipette aspiration of living cells. J Biomech. 33 (1), 15-22 (2000).
  21. Vajta, G., et al. Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the handmade cloning (HMC) and the submarine incubation system (SIS). Theriogenology. 62 (8), 1465-1472 (2004).
  22. Alteri, A., et al. Revisiting embryo assisted hatching approaches: A systematic review of the current protocols. J Assist Reprod Genet. 35 (3), 367-391 (2018).
  23. Briski, O., Salamone, D. F. Past, present and future of ICSI in livestock species. Anim Reprod Sci. 246, 106925(2022).
  24. Wall, R. J. Pronuclear microinjection. Cloning Stem Cells. 3 (4), 209-220 (2001).
  25. Selokar, N. L., et al. Hope for restoration of dead valuable bulls through cloning using donor somatic cells isolated from cryopreserved semen. PLoS One. 9 (3), e90755(2014).
  26. Selokar, N. L., et al. Buffalo (Bubalus bubalis) SCNT embryos produced from somatic cells isolated from frozen-thawed semen: Effect of trichostatin A on the in vitro and in vivo developmental potential, quality and epigenetic status. Zygote. 24 (4), 549-553 (2016).
  27. Guevorkian, K., Maître, J. L. Micropipette aspiration: A unique tool for exploring cell and tissue mechanics in vivo. Methods Cell Biol. 139, 187-201 (2017).
  28. Duah, E. K., et al. Production of hand-made cloned buffalo (Bubalus bubalis) embryos from nonviable somatic cells. In Vitro Cell Dev Biol Anim. 52 (10), 983-988 (2016).
  29. Loi, P., et al. Nuclei of nonviable ovine somatic cells develop into lambs after nuclear transplantation. Biol Reprod. 67 (1), 126-132 (2002).
  30. Fahrudin, M., Otoi, T., Suzuki, T. Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues. J Vet Med Sci. 63 (10), 1151-1154 (2001).
  31. Podico, G., et al. Single-layer colloid centrifugation as a method to process urine-contaminated stallion semen after freezing-thawing. J Equine Vet Sci. 87, 102910(2020).
  32. Zhang, Y. T., et al. Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer. J Vet Sci. 23 (2), e40(2022).
  33. Mizutani, E., et al. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells. Sci Rep. 6, 23808(2016).
  34. Long, S., Kenworthy, S. Round cells in diagnostic semen analysis: a guide for laboratories and clinicians. Br J Biomed Sci. 79, 10129(2022).
  35. Johanisson, E., Campana, A., Luthi, R., de Agostini, A. Evaluation of 'round cells' in semen analysis: a comparative study. Hum Reprod Update. 6 (4), 404-412 (2000).
  36. Graber, M., Lane, B., Lamia, R., Pastoriza-Munoz, E. Bubble cells: Renal tubular cells in the urinary sediment with characteristics of viability. J Am Soc Nephrol. 1 (7), 999-1004 (1991).
  37. Rahmanian, N., Bozorgmehr, M., Torabi, M., Akbari, A., Zarnani, A. H. Cell separation: Potentials and pitfalls. Prep Biochem Biotechnol. 47 (1), 38-51 (2017).
  38. Håård, M. Use of frozen stallion semen in Europe. Acta Vet Scand Suppl. 88, 123-127 (1992).
  39. Klug, E., Treu, H., Hillmann, H., Heinze, H. Results of insemination of mares with fresh and frozen stallion semen. J Reprod Fertil Suppl. 23, 107-110 (1975).
  40. Pukazhenthi, B., Comizzoli, P., Travis, A. J., Wildt, D. E. Applications of emerging technologies to the study and conservation of threatened and endangered species. Reprod Fertil Dev. 18 (1-2), 77-90 (2006).
  41. Swanson, W. F., Magarey, G. M., Herrick, J. R. Sperm cryopreservation in endangered felids: developing linkage of in situ-ex situ populations. Soc Reprod Fertil Suppl. 65, 417-432 (2007).
  42. Lagutina, I., Fulka, H., Lazzari, G., Galli, C. Interspecies somatic cell nuclear transfer: Advancements and problems. Cell Reprogram. 15 (5), 374-384 (2013).

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Somatic Cell IsolationNuclear Donor CellsSomatic Cell Nuclear TransferEquine CloningCell Morphology SelectionMicropipette AspirationSequential Cell WashingDiploid Cell Recovery
A Simple Microaspiration Technique for Isolating Somatic Cells from Cryopreserved Equine Semen as Nuclear Donors for Cloning
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