Biology Reference
In-Depth Information
3.3.2 Impact of Classical and Emerging
Preservation Techniques
Spermatozoa are commonly exposed to a variety of perturbations for the purposes
of evaluation, processing, freezing, and practical use. Classical freeze-preservation
approaches that rely on exposing these cells to cryoprotectant, osmotic, and
cooling/freezing/thawing stressors are well known to adversely affect the quality
of cat spermatozoa (Pukazhenthi et al.
2006
). However, the use of a slow freezing
(circa -1C/min) method has been shown to retain both normal centrosomal
structure and function (Comizzoli et al.
2006
; Table
3.1
). There has been growing
interest in the scientific community in preserving male gametes via freeze-drying,
desiccation, or in a liquid environment at supra-zero temperatures. While prom-
ising, these methods easily compromise sperm motility, thereby complicating the
ability to select spermatozoa that have functional centrosomes. Interestingly,
freeze-drying apparently is not detrimental to centrosomal functions of non-human
primate (Sánchez-Partida et al.
2008
) and bovine (Hara et al.
2011
) spermatozoa.
However, primate spermatozoa that are simply desiccated in trehalose appear to
lose fertilization potential (Klooster et al.
2011
). These important and contradic-
tory findings deserve more thorough validation, especially as Ringleb et al. (
2011
)
recently determined that the injection of freeze-dried cat spermatozoa into oocytes
leads to limited early embryo development. We have also confirmed the com-
promised sperm aster formation using desiccated spermatozoa (dried at ambient
temperature in trehalose) that were injected into conspecific oocytes (Table
3.1
).
We suspect that both phenomena are due to altered centrosomal functions. One
study has also demonstrated loss in centrosomal capacity after storing cat sper-
matozoa in alcohol (Murakami et al.
2005
). However, recently, we have effectively
preserved cooled (4 C) cat spermatozoa for up to 2 weeks in a 2 M trehalose
solution while successfully retaining DNA integrity, centrosomal structure (pres-
ence of centrin), and function (sperm aster formation; Table
3.1
).
3.4 Mitigating Centrosomal Immaturity and Dysfunctions
3.4.1 Sperm Micromanipulations and Selections
It is possible to use sonication and micromanipulation to replace an immature
centrosome from a non-functioning testicular spermatozoon with a mature coun-
terpart from an ejaculated cell. These reconstructed cat spermatozoa have bio-
logical viability, at least in capacity to develop into blastocysts in vitro (Comizzoli
et al.
2006
). Interestingly, such re-built spermatozoa are comprised of a head and
centrosome/midpiece that are sufficiently proximate to ensure adequate interac-
tion, pronuclear alignment, and linear migration no different from that observed
after microinjecting an intact (un-reconstructed) spermatozoon (Van Blerkom and
