El resumen de la tesis es el siguiente:
Due to the increasing number of zebrafish (Danio rerio) mutant and transgenic lines, there is a high demand for assisted reproductive techniques to support facility management. Efficient zebrafish sperm cryopreservation is a pressing necessity to manage and preserve the valuable zebrafish genetic resources.
Although zebrafish sperm
cryopreservation was first attempted more than 30 years ago, protocols still
lack standardization, which translates into high variability in post-thaw sperm
quality and in vitro fertilization success. Therefore, the present thesis aims
to improve the current methodologies used for zebrafish sperm cryopreservation
and broodstock management towards the standardization of procedures in this
species.
The introductory context
of the present thesis is approached in chapter 1. In this chapter the relevance
of zebrafish model is discussed as well as this species sperm cryopreservation
usefulness. The main factors affecting sperm quality and the application of
reliable quality analysis are discussed in this chapter. The final objective of
sperm cryopreservation is to obtain high quality offspring and therefore in
vitro fertilization, early development and offspring quality analysis are
important tools for the optimization of sperm cryopreservation methodologies.
The current knowledge in sperm cryopreservation fundamentals is approached in
this chapter, as well as the main advances and bottlenecks in zebrafish sperm
cryopreservation.
In chapter 2, the
zebrafish sperm motility activation was assessed under different conditions of
water temperature and conductivity. The environmental conditions present in the
fertilization microenvironment are responsible for the mechanism of spermatozoa
motility activation and metabolic modulation that influence the probability of
fertilization success. Zebrafish is commonly reared at 28°C, but with variable
water conductivity conditions among facilities. However, sperm motility
analysis is routinely performed with distilled water at room temperature. We
aimed to understand the effect of water temperature and conductivity on sperm
motility and fertilization ability. Water at 28°C with lower water conductivity
(0 and 700 μS/cm) improve sperm motility parameters.
Standardization of the
water conditions (of system water and activation medium used for motility
analysis) among facilities is highly relevant to improve the reproducibility of
sperm quality analysis and thus, to predict with higher accuracy fertilization
ability.
Successful
cryopreservation depends on high quality sperm, which depends on the quality of
breeders. Consequently, broodstock selection and management is a priority to
improve sperm cryopreservation. The broodstock diet has a preponderant effect
on gamete quality, particularly in phospholipids and antioxidants content which
are known to promote spermatogenesis. Therefore, in chapter 3 we aimed to
determine the effects of a tailor-made purified diet supplemented with
phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the zebrafish
reproductive performance, gamete quality and larval skeletal malformations.
Both dietary supplementations with phospholipids improved sperm motility and
eggs quality, however PC increased the incidence of skeletal malformations on
the offspring, as previously observed in other teleosts. Although dietary
phospholipids classes have a role in the ossification process of the vertebral column
in teleosts, its mechanisms are still to be understood. Therefore, the development
and use of a standardized diet for zebrafish broodstock is essential to reduce
the variability of the reproductive performance among facilities. In chapter 4,
the selection of optimal age and minimum sperm collection frequency was evaluated,
since these factors are essential to obtain high quality samples. Our results
indicate that young males (6-8 months) showed higher sperm quality and require
a minimum of 14 days between sperm collections to recover sperm plasma membrane
viability.
An important bottleneck in
cryopreservation is the liquid nitrogen requirement for storage. Therefore, it
was established in chapter 5 a new cryopreservation method using an electric
ultrafreezer (-150°C) as an alternative to liquid nitrogen, for the first time
in a teleost species. This protocol reaches a fast cooling rate (-66°C/min) in
one single step and yields higher sperm viability and DNA integrity in
comparison to the traditional methods (-20°C/min in liquid nitrogen). The
synergy obtained by the combination of cryoprotectants is a successful
cryopreservation strategy that can be beneficial in the optimization of zebrafish
sperm cryopreservation. Therefore, it was selected the most adequate cryoprotectant
combination that generates offspring with normal skeletogenesis. Data show that
15% of DMF with 50 mM of bicine or 10% of egg yolk is beneficial for the
quality of zebrafish offspring sired by cryopreserved sperm. To the best of our
knowledge, this is the first report on skeletal development of zebrafish
offspring sired by cryopreserved sperm performed with different extender
compositions.
Zebrafish is especially
useful to investigate some of the most prominent human diseases such as
diabetes. Among other consequences, diabetes (type I and II) causes
disturbances in the male reproductive system, since glucose metabolism is an
important event not only in spermatogenesis but also in mature spermatozoa metabolism.
In chapter 6 we aimed to validate zebrafish as a useful model organism to
investigate male reproductive dysfunctions mechanisms caused by type I diabetes.
In this chapter, sperm cryopreservation was applied to a relevant zebrafish
model of type I diabetes. The transgenic zebrafish under diabetic conditions
shows higher levels of insulin a (insa), insulin receptor a (inra) and glucose
carrier 2 (slc2a2) transcripts in spermatozoa when compared to the controls.
This is because gametogenesis occurred under diabetic conditions, changing
transcription in the germline. Consequently, spermatozoa carry the imprinted
transcripts that will be transmitted during fertilization. Sperm quality (motility,
viability and DNA integrity) was lower in the transgenic fish under (transient)
diabetic state as observed in human and mouse model. Sperm cryopreservation
affects sperm quality of fish both under diabetic and non-diabetic conditions.
However, diabetic conditions were detrimental in sperm freezability, which can
be explained by the lower initial sperm quality. In this chapter zebrafish was
validated as a useful model organism to investigate male reproductive dysfunctions
mechanisms caused by type I diabetes.
Relevant differences between different zebrafish lines are evidenced in
terms of sperm quality and susceptibility to damage, which suggests that it is
an important factor to consider while establishing sperm cryopreservation
protocols. This thesis offers new insights and a set of guidelines on breeder’s
management and sperm cryopreservation to improve zebrafish husbandry practices.
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