Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

Journal Pre-proof Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis Patrícia Diogo, Gil Martins, Rita Nogueira, Ana Marreiros, Paulo J. Gavaia, Elsa Cabrita PII:

S0011-2240(19)30169-5

DOI:

https://doi.org/10.1016/j.cryobiol.2019.10.001

Reference:

YCRYO 4124

To appear in:

Cryobiology

Received Date: 6 June 2019 Revised Date:

6 September 2019

Accepted Date: 3 October 2019

Please cite this article as: Patrí. Diogo, G. Martins, R. Nogueira, A. Marreiros, P.J. Gavaia, E. Cabrita, Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis, Cryobiology (2019), doi: https://doi.org/10.1016/j.cryobiol.2019.10.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

BSA

Egg Yolk

Glycine Bicine

1

Cryoprotectants synergy improve zebrafish sperm cryopreservation and

2

offspring skeletogenesis

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Patrícia Diogo1, Gil Martins1, Rita Nogueira1, Ana Marreiros2,3, Paulo J. Gavaia1,2,

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Elsa Cabrita1*

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1

Centre of Marine Sciences, University of Algarve, 8005-139 Faro, Portugal.

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2

Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139

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Faro, Portugal.

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3

Algarve Biomedical Center, Campus Gambelas, 8005-139 Faro, Portugal.

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*Corresponding author: Tel.: +351.289 800532; Fax: +351.289 800 069. E-mail

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address: [email protected]

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Abstract

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The synergy obtained by the combination of cryoprotectants is a successful strategy

17

that can be beneficial on the optimization of zebrafish sperm cryopreservation.

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Recently, a protocol was established for this species using an electric ultrafreezer (-

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150 ºC) performing cooling rate (-66 ºC/min) and storage within one step. The

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ultimate objective of sperm cryopreservation is to generate healthy offspring.

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Therefore, the objective of this study was to select the most adequate cryoprotectant

22

combination, for the previously established protocol, that generate high quality

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offspring with normal skeletogenesis. Among the permeating cryoprotectant

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concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded

25

high post-thaw sperm quality and hatching rates. For these two concentrations, the

1

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presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM)

27

and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro

28

fertilization success and offspring skeletal development (30 days post fertilization).

29

Higher concentration of permeating cryoprotectant (15%) decreased the incidence of

30

deformed arches and severe skeletal malformations, which suggests higher capacity

31

to protect the cell against cold stress and DNA damage. Extender containing 15%

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DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with

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higher post-thaw quality. The use of these compounds results in a reduction in

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vertebral fusions, compressions and severity of skeletal malformations in the

35

offspring. Therefore, these extender compositions are beneficial for the quality of

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zebrafish offspring sired by cryopreserved sperm with -66 ºC/min freezing rate. To

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the best of our knowledge, this is the first report on skeletal development of the

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offspring sired by cryopreserved sperm performed with different freezing media

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compositions in zebrafish.

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Keywords: Cryopreservation, zebrafish sperm, offspring, skeletal malformations,

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decision trees, cryoprotectants

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Introduction

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Zebrafish is the second most used model organism with increasing interest by the

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scientific community in the past decade. Consequently, new mutant and transgenic

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lines are developed continuously in laboratories across the world, posing challenges

48

in terms of space and management that cryopreservation can solve. Until today,

49

zebrafish sperm cryopreservation lacks standardization, yielding variable post-thaw

50

sperm quality and in vitro fertilization success [3, 64, 75]. Recently, our laboratory

2

51

developed the first cryopreservation protocol in a teleost species using an electric

52

ultrafreezer (-150 ºC). This protocol does not require liquid nitrogen or dry ice,

53

samples are placed directly on the electric ultrafreezer where the freezing rate (-66

54

ºC/min) and storage occurs in one single step [25]. The use of ultrafreezers for sperm

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cryopreservation and storage allow the reduction of the global costs of

56

cryopreservation and simplify the procedure. Therefore, it is a valuable alternative

57

cryopreservation method for zebrafish facilities management. Following the

58

establishment of this protocol, the present work aims to optimize the freezing

59

medium by modulating the permeating and non-permeating cryoprotectants

60

composition.

61

A cryoprotectant agent is a solute that when present in the cells medium, allow

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higher post-thaw recoveries than if it is not present [42]. Cryoprotectants are

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categorized as permeating and non-permeating, according to their ability to penetrate

64

cellular membranes [22, 28]. In cryobiology, it has become clear that distinct

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cryoprotectant classes can efficiently protect cells against freezing injuries through

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multiple mechanisms, many of which are still poorly understood [28, 50]. The

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combination of permeating and non-permeating cryoprotectants is considered a

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successful strategy [28] widely used among sperm cryopreservation protocols of

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teleost species [14].

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The protocol developed in our facilities for zebrafish sperm cryopreservation [25]

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comprises a freezing medium with 10% of N,N-dimethylformamide (DMF) in

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Hank´s balanced salt solution (HBSS) for a -66 ºC/min freezing rate. This method

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improved post-thaw sperm DNA integrity, plasma membrane viability and late

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apoptosis (detection of disrupted plasma membrane and phosphatidylserine

75

externalization) [25]. The permeating cryoprotectant concentration was previously

3

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selected for slower cooling rates performed in dry ice [2, 25]. Considering that cell

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biophysical properties vary with temperature [28], it was essential to investigate the

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most appropriate concentration of DMF for a -66 ºC/min freezing rate, to improve

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the previously established protocol. In other teleost species similarly fast cooling

80

rates improved post-thaw sperm quality [6, 7]. There are structural, morphological

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and biophysical similarities observed between spermatozoa of zebrafish and other

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cyprinid species [80]. These facts suggest that methodological improvements for

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cryopreservation in cyprinid species may benefit zebrafish sperm post-thaw quality.

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In cyprinids, freezing media commonly contain bicine and glycine [14, 34, 78],

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therefore it was pertinent to investigate the effect of these compounds in zebrafish

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sperm cryopreservation. Additionally, Bovine Serum Albumin (BSA) and Egg Yolk

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(EY) were selected as non-permeating cryoprotectants due to their extensive use in

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cryopreservation of sperm from several species, with beneficial post-thaw outcomes

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[14, 58, 63].

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Sperm fertilizing ability is considered the most effective quality analysis to validate

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the effectiveness of a sperm cryopreservation protocol [14, 31, 65]. However, the

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quality of the offspring generated by cryopreserved sperm beyond hatching rate have

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been poorly addressed [27, 44, 56, 59, 76, 79], particularly the incidence of

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malformations [56, 79] which were only studied immediately after hatching. Since

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skeletal development and incidence of malformations is a well-established fish

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quality evaluation system [10, 11], it is a useful tool for the characterization of

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offspring quality sired by cryopreserved sperm.

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The description of skeletal malformations generates complex data sets with high

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biological variability, being therefore difficult to analyze in depth through traditional

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statistical methods. Machine learning is a method focused on the development of

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algorithms that are particularly useful for data mining. These algorithms are able to

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automatically learn to recognize complex patterns based on data. Classification or

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decision trees are machine learning methods that can provide guidelines for decision

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making [12]. Decision trees are non-parametric models that use algorithms to split

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data sets into increasingly homogeneous subsets, representing class membership

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through hierarchal distribution. Therefore, classification trees are considered a

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“knowledge discovery” technique [20], which have been considered a powerful tool

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for the optimization of cryopreservation technologies [61, 66], although it is still

109

poorly explored. This modeling technique is flexible enough to handle complex

110

problems with multiple interacting elements, yielding a straightforward

111

interpretation [20]. Consequently, it is an ideal method to explore the effects of

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cryoprotectant combinations during zebrafish sperm cryopreservation on the

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resulting offspring skeletogenesis.

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The objective of this study was to select the optimal combination of permeating and

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non-permeating cryoprotectants for zebrafish sperm cryopreservation, performed

116

with an electric ultrafreezer (-66 ºC/min freezing rate). For that purpose, the effect of

117

permeating cryoprotectant (DMF) concentration on post-thaw sperm quality and in

118

vitro fertilization was investigated. Additionally, the interactions between the

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combinations of two concentrations of the permeating cryoprotectant (12.5% and

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15% DMF) and the addition of non-permeating cryoprotectants (10 mg/mL of BSA,

121

10% of EY, 30 mM glycine and 50 mM of bicine) were evaluated. Finally, the

122

skeletal malformations of the offspring sired by sperm cryopreserved with different

123

freezing media compositions was studied for the first time, to select the protocol

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which generated offspring with the higher skeletal quality.

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Material and methods

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Fish rearing

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Adult AB zebrafish males and females were selected according to the age selection

129

criteria previously established in our laboratory (6-8 months old) [26]. Zebrafish

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with similar size were maintained separated according to sex into 3.5 L tanks (n =

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15) to improve fecundity, egg viability and early larvae survival [43]. The fish were

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maintained in a water recirculation system (ZebTEC® Tecniplast, Italy). The fish

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room had a controlled photoperiod with a 14:10 h light/dark cycle, an independent

134

air conditioning system (26 ± 1 °C) and an air extraction system to guarantee the air

135

renewal in the room, maintaining the humidity close to 60%. The water rearing

136

system was partially replaced (10%) daily and the water system maintained at 28.5 ±

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0.5 °C, 700 ± 50 µS and pH 7.5 ± 0.1. The fish were fed ad libitum twice a day with

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Artemia nauplii (AF480, INVE, Belgium) and ZEBRAFEED® diet (Sparos Lda,

139

Portugal). Food consumption was visually controlled, and the remains removed

140

daily. All animal manipulations were performed in compliance with the Guidelines

141

of the European Union Council (86/609/EU) and transposed to the Portuguese law

142

for the use of laboratory animals on research by “Decreto Lei n° 129/92 de 06 de

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Julho, Portaria n° 1005/92 de 23 de Outubro”, and according to the European

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parliament council directive´s for protection of animals used for scientific research

145

(2010/63/EU). All animal protocols were performed under a “Coordinator-

146

researcher” license from the Direção-Geral de Veterinária, Ministério da Agricultura,

147

do Desenvolvimento Rural e das Pescas, Lisbon, Portugal, under the “Decreto Lei

148

n°113/2013 de 7 de Agosto” relative to the protection of animals used for scientific

149

research.

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Sperm collection

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On the day prior to the sperm collection, males (n = 4) and females (n = 4) were

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placed in 1 L breeding tanks (Tecniplast, Buguggiate, Italy) and maintained

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separated while sharing the same water for 16 h [25]. This method allows the

155

exposure to the pheromones of the mating partners, which promotes the

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synchronization of mating behavior and oocyte release [33, 68, 69]. Sperm collection

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was performed, within 1 h after the beginning of the light phase of the photoperiod.

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Males were properly anesthetized with 0.168 mg ml-1 tricaine sulfonate solution

159

(MS-222, Sigma-Aldrich, Madrid, Spain) according to Westerfield [73]. When the

160

gill movement decreased, the males were rinsed with Phosphate Buffered Saline

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(PBS) solution and carefully cleaned with paper towels. For sperm collection, an

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abdominal massage was performed and the sperm collected using a glass capillary

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tube attached to a mouth piece. Immediately after collection, sperm was diluted into

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10 µl of sterilized and filtered (0.20 µm) Hank´s Balanced Salt Solution (HBSS) at

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300 mOsm/Kg (NaCl 8.0 g, KCl 0.4 g, CaCl2 x 2H2O 0.16 g, MgSO4 x 7H2O 0.2 g,

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Na2HPO4 0.06 g, KH2PO4 0.06 g, NaHCO3 0.35 g, C6H12O6 1.0 g in 1000 mL of

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milli-Q water, pH 7.5) [36, 41].

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Experiment 1 – Permeating cryoprotectant: DMF concentration

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An experiment was conducted to evaluate the adequate N-N dimethylformamide

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(DMF) concentration necessary to protect spermatozoa from cold damage using a -

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66 ºC/min freezing rate in an electric MDF-C2156VAN ultra-low temperature

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freezer (Sanyo, Demark). To perform sperm pools (n = 6 pools) for this experiment,

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we selected sperm samples from males with total motility over 50% (at 10 s post

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activation) and cell concentration over 3 x 107 cells/mL. Each sperm pool contained

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sperm from 10 males. A cooling rate of -66 ºC/min was applied placing the cryovials

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with sperm diluted in the freezing medium directly in an ultrafreezer system (-150

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ºC) as previously described [25]. Sperm was cryopreserved with 5, 7.5, 10, 12.5 or

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15% of DMF in HBSS in a final volume of 10 µL (of prediluted sperm added to

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freezing medium) and stored in 2 ml cryovials (VWR® Low Temperature Freezer

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Vials). After 5 days of storage in the ultrafreezer system, thawing was performed in a

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40 ºC water bath during 8 s. Sperm quality was evaluated through sperm motility,

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membrane integrity and in vitro fertilization success.

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Experiment 2 – Non-permeating cryoprotectants: BSA, egg yolk, glycine and bicine

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According to the results obtained in experiment 1, 12.5 and 15% DMF were selected

187

to evaluate the effect of non-permeating cryoprotectants on the freezing medium

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used for zebrafish sperm cryopreservation. For each DMF concentration a control

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(Ctrl) without non-permeating cryoprotectant was used and it was evaluated the

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effect of 10 mg/mL BSA (BSA), 10% egg yolk (EY), 30 mM glycine (Gly) and 50

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mM of bicine (Bici) on the freezing medium. The concentrations of non-permeating

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cryoprotectants were chosen according to the commonly used in other successful

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sperm cryopreservation protocols for teleost species [39, 49, 53, 63]. To characterize

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post-thaw sperm quality (n = 5 pools, each pool containing sperm of 16 males), the

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evaluation of sperm motility, plasma membrane viability and in vitro fertilization

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success were performed.

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Sperm concentration and motility analysis

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Sperm concentration and motility was evaluated using computer assisted sperm

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analysis (CASA) system (ISAS Integrated System for Semen Analysis, Proiser,

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Valencia, Spain) coupled to a phase contrast microscope (Nikon E-200, Nikon,

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Tokyo, Japan) with a ×10 negative phase contrast objective. The images were

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captured with ISAS 782C camera (Proiser, Spain) and processed with CASA

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software. The settings of the CASA system were adapted previously for this species

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namely 25 frames/s, connectivity 14, 1 to 90 mm for head area and only sperm

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samples with VCL > 10 µm/s were considered motile. For sperm concentration a

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dilution (1:19) was performed with HBSS and 3 fields were sampled to determine

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cell concentration. For motility analysis 0.5 µL of fresh sperm or 1.5 µL of

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cryopreserved sperm was placed on a Mackler chamber and immediately activated

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with 5 µL of filtered (0.20 µm) and sterilized system water at 28 ºC. Sperm motility

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was characterized at 10 s post-activation according to total motility (TM; %),

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progressive motility (PM; %), curvilinear velocity (VCL; µm/s), straight-line

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velocity (VSL; µm/s) and linearity (LIN; %).

214 215

Membrane integrity

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Sperm membrane integrity was assessed through flow cytometry using SYBR 14

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(Invitrogen, Spain) and Propidium Iodide (PI) (Sigma Aldrich, Spain) labelling,

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according to previously established methodology [25]. SYBR 14 is a permeant

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nucleic acid stain that crosses plasma membrane and PI is a membrane impermeable

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dye that label cells nucleic acids when the plasma membrane is compromised.

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Consequently, spermatozoa with compromised plasma membrane are labelled in red

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from PI and viable cells are labelled in green by SYBR 14 [21]. SYBR 14 was

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prepared diluting 5 µL of stock solution in 120 µL of sterilized and filtered HBSS,

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and PI was used undiluted. The pre-diluted sperm samples were re-diluted (1:300) in

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HBSS and each stain was added in a final concentration of 6.7 nM of SYBR 14 and

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3 ng/mL of PI. Sperm was incubated for 5 min in the dark at room temperature. The

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analysis was performed in a flow cytometer (BD FACSCalibur™, BD Biosciences,

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Spain) adjusted for the detection of SYBR 14 through a 530 nm bandpass filter

229

(FL1) and PI was detected with a 670 nm long pass filter (FL3). Flow cytometer

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settings were previously adjusted using a positive (100% dead cells) and a negative

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control (fresh sperm). As negative control, spermatozoa were exposed to successive

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cycles of freezing thawing [13]. A total of 5000-10000 events were counted for each

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sample.

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In vitro fertilization

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Females used for in vitro fertilization were maintained in a breeding tank separated

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from males for 16 h previously to the experiments. Females were anesthetized with

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MS-222, as described above. When the gill movement decreased, the females were

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rinsed with sterile PBS (pH 7.4) and placed in a 35 mm Petri dish. An abdominal

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massage was carefully performed to collect an aggregate of oocytes (clutch),

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avoiding any mechanical contact. If the clutch of oocytes had good quality

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characteristics, namely no broken or white eggs and have a hyaline and yellowish

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color [9, 16], in vitro fertilization was performed within 1 min after collection. Only

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good quality clutches were selected both for experiment 1 (n = 20) and experiment 2

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(n = 24). Each clutch was used for one in vitro fertilization. Each sperm pool from

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each experimental condition (fresh and cryopreserved) was used to fertilize 2-5

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clutches of different females to reduce the maternal derived effects (Fig 1). This

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methodology allowed to avoid oocyte manipulation which produces embryo abortion

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24 hpf.

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The sperm contained in one cryovial was used for each fertilization. Therefore, a

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total of 1.5 - 2 x 106 spermatozoa was added to the oocytes (100-200) of either fresh

252

or thawed samples. Sperm motility activation was immediately performed with 360

253

µL of sterilized and filtered (0.2 µm) system water at 28 ºC. After 5 min, 5 mL of

254

system water was added to the Petri dish containing the eggs. The embryos were

255

maintained in an incubator at 28 ºC with the same photoperiod as in the zebrafish

256

facilities (14L:10D). All the dead embryos were removed, and the viable embryos

257

transferred to 100 mm Petri dishes 3 - 4 hours post fertilization (hpf). Survival and

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hatching rates were calculated at 24 and 72 hpf, according to the initial number of

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oocytes of each clutch (approximately 100-200). For each treatment, each sperm

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pool was used to fertilize 2-5 clutches of eggs.

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Skeletal development analysis of the offspring obtained from both experiences

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The evaluation of skeletal malformations in the offspring generated by cryopreserved

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sperm in both experiments, was performed in zebrafish juveniles at 30 days post

265

fertilization (dpf). The fish were anesthetized with a lethal dose of MS-222 (300

266

mg/mL) (Sigma-Aldrich, Saint Louis, MO) [52] and fixed in a 4% buffered

267

paraformaldehyde solution at 4 °C for 24 h. Juvenile zebrafish were further washed

268

with PBS, pH 7.4 and stored in 75% ethanol at room temperature [32]. A modified

269

method of whole-mount acid-free double staining was performed using alcian blue

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8GX (Sigma-Aldrich, Saint Louis, MO) for cartilage and alizarin red S (Sigma-

271

Aldrich, Saint Louis, MO) for bone [70]. Briefly, samples were stained in alcian blue

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8GX for 1.5 h and passed through a decreasing series of ethanol concentrations (96

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to 25%) and hydrated with distilled water before being stained with alizarin red S in

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a potassium hydroxide solution 0.5% overnight. The samples were cleared with a

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0.5% KOH solution and stored in a solution of 90% glycerol (Merk Millipore,

276

Billerica, MA) at room temperature. Zebrafish juveniles at 30 days post fertilization

277

have completed the skeletal structures mineralization and the detection of skeletal

278

anomalies was performed following the nomenclature by Bird and Mabee [8].

279

Briefly, the description of skeleton malformations was performed for each skeleton

280

structure namely arches (neural and heamal) and centra (vertebrae) distributed within

281

each region of the axial skeleton (Weberian apparatus, precaudal vertebrae, caudal

282

vertebrae and caudal fin vertebrae). When the same individual had multiple skeletal

283

malformations in the same region, they were all considered in the data used to

284

investigate the charge of malformations observed. The severe malformations

285

considered were lordosis (V-shaped dorsal–ventral curvature), kyphosis (Λ-shaped

286

dorsal–ventral curvature) and scoliosis (lateral curvature). The occurrence of fusions,

287

compressions, abnormal arches, extra arches, opened arches and deformed centra

288

were evaluated and images acquired with a stereomicroscope SteREO Lumar.V12

289

(Zeiss, Germany).

290 291

Data analysis

292

IBM SPSS Statistics 25.0 software was used for statistical analysis. Data were

293

expressed as means ± SD (Standard Deviation) and normalized by logarithmic, or

294

arcsine transformation when results were expressed as percentages. In experiment 1

295

to evaluate the significance of the permeating cryoprotectant concentration effect on

296

post-thaw sperm quality and in vitro fertilization success, a one-way ANOVA

297

multiple comparison test (Student–Newman–Keuls, P < 0.05) was performed. In

298

experiment 2, significant differences between fresh and cryopreserved sperm were

299

detected through independent samples t-test (P < 0.05) and differences between

12

300

permeating cryoprotectant concentration and non-permeating cryoprotectants were

301

detected through a two-way ANOVA, with Student–Newman–Keuls post hoc to

302

evaluate differences between non-permeating cryoprotectants and independent

303

samples t-test to evaluate the effect of cryoprotectant concentration (P < 0.05).

304

The incidence of malformations, their severity, distribution and load per fish

305

obtained by each treatment was investigated through Pearson´s Chi-square analysis

306

(P < 0.05).

307

For a deeper comprehension of the relationship between cryoprotectant composition

308

during zebrafish sperm cryopreservation and the onset of skeletal malformations on

309

the resulting offspring, a machine learning technique was performed, complementing

310

the traditional statistical analysis. Since the variables are potentially correlated with

311

each other a decision tree was applied through the algorithm CART (classification

312

and regression), that uses GINI index splicing criteria (a measure of statistical

313

dispersion). These tree models classify cases into groups or predict values of a

314

dependent variable (criterion), based on values or categories of the independent

315

variables (predictors). The criterion used was the classification of fish having either

316

malformed or normal axial skeleton in relation to the following factors: permeating

317

cryoprotector concentration, non-permeating cryoprotector, treatment (combination

318

of cryoprotectants), vertebral compression, fusions, additional arches, opened arches,

319

deformed arches, deformed centra and number of load of deformations. A maximum

320

tree depth of 5 levels was generated by the algorithm with a minimum number of 20

321

initial (parent) nodes and 10 terminal (child) nodes, able to differentiate groups with

322

normal and malformed individuals (dependent variable in node 0). Variables were

323

not considered in the decision tree if a regression could not be generated by the

324

algorithm, therefore no homogenous groups were formed and the variable was not

13

325

represented in the decision tree (e.g. opened arches). Therefore, when the algorithm

326

is able to generate a new level of ramification from a node, means that the new

327

groups formed have significantly differently normal or malformed individuals.

328 329

Results

330

Permeating cryoprotectant concentration

331

The effect of different permeating cryoprotectant (DMF) concentrations on sperm

332

quality and in vitro fertilization success displayed a normal distribution on the

333

analysis of total motility, progressive motility, embryo survival at 24 hpf and

334

hatching rate (Fig 2). These parameters were more representative of the effect of

335

permeating cryoprotectant concentration when compared to sperm velocities and

336

linearity.

337

Post-thaw sperm quality was significantly lower than fresh sperm in terms of total

338

motility, progressive motility and plasma membrane viability (Fig 2A, B and F).

339

Freezing media containing 12.5 and 15% of DMF showed significantly higher post-

340

thaw sperm total motility and plasma membrane viability (Fig 2B and F).

341

Additionally, these treatments showed no significant differences when compared to

342

fresh sperm curvilinear and straight-line velocity (Fig 2C and D). Linearity was

343

affected in cryopreserved sperm with 5 and 10% of DMF when compared to fresh

344

sperm and sperm cryopreserved with 12.5 and 15% of DMF (Fig 2E).

345

The use of 5% of DMF yielded significantly lower embryo survival when compared

346

to fresh sperm and the other cryopreserved treatments (Fig 2G). The hatching rate

347

was significantly higher in fresh sperm and sperm cryopreserved with 10 and 12.5%

348

of DMF when compared to 5%, however both concentrations showed no significant

14

349

differences when compared to 7.5 and 15% of DMF (Fig 2H). The treatment that

350

yielded lower post-thaw sperm quality was 5% of DMF.

351

Considering the overall sperm quality and in vitro fertilization analysis, 12.5% and

352

15% of DMF were selected to investigate the effect of non-permeating

353

cryoprotectants on post-thaw sperm quality and offspring skeletal development.

354 355

Non-Permeating cryoprotectants

356

The post-thaw sperm quality and in vitro fertilization parameters analyzed did not

357

show significant interactions between main factors (permeating*non-permeating)

358

(two-way ANOVA, P < 0.05), except for total motility (Table 1). The lack of

359

interactions (except in total motility) allow the direct interpretation of the main

360

treatments effects independently. Consequently, the effect of permeating

361

cryoprotectant concentration and non-permeating cryoprotectants can be studied

362

independently, except in total motility. Progressive motility, velocities and linearity

363

were significantly dependent on the presence of non-permeating cryoprotectants, but

364

not on the permeating cryoprotectant concentration. Plasma membrane viability was

365

significantly dependent of both permeating cryoprotectant concentration and non-

366

permeating cryoprotectants addition (Table 1).

367

Fresh sperm had significantly higher total and progressive motility, velocities,

368

linearity and plasma membrane viability when compared to cryopreserved sperm.

369

However, the in vitro fertilization parameters were not significantly different from

370

cryopreserved sperm (Fig 3). Despite the fact that BSA with 12.5% of DMF yielded

371

high sperm total motility, this treatment showed a relevant impairment of

372

progressive movement, plasma membrane integrity and in vitro fertilization success.

15

373

Overall, the use of BSA had a negative effect on post-thaw sperm quality, especially

374

in sperm progressive motility and plasma membrane viability (Fig 3A, B and F).

375

The use of egg yolk as non-permeating cryoprotectant in the freezing medium with

376

15% DMF improved significantly sperm total motility when compared to the other

377

treatments, except control (Fig 3A).

378

The addition of bicine on the freezing medium composition significantly improved

379

progressive motility when compared to BSA (Fig 3B). Both velocities were

380

significantly improved in samples submitted to control treatment when compared to

381

BSA, however control velocities were not significantly different to egg yolk and

382

glycine treatments (Fig 3C and D). Spermatozoa linearity movement was

383

significantly higher in control treatment when compared to BSA, but not to the other

384

non-permeating cryoprotectants (Fig 3E).

385

The plasma membrane viability of control and bicine treatment was significantly

386

higher than BSA, but there were no differences when compared to egg yolk and

387

glycine treatment (Fig 3F). There were no statistical differences in in vitro

388

fertilization parameters namely embryo survival and hatching rates (Fig 3G and H).

389

Although males were thoroughly selected sample quality thresholds established as

390

previously described, the in vitro fertilization success was highly variable which can

391

be observed in the standard error bars in Figure 2G and H, 3G and H and in

392

supplementary tables 1, 2 and 3.

393 394

Axial Skeleton malformations

395

The characterization of severe skeletal malformations was more conclusive in terms

396

of differences between permeating cryoprotectant percentage, in comparison to the

397

total percentage of skeletal malformations incidence on the offspring sired by

16

398

cryopreserved sperm (Fig 4A and B). The percentage of deformities observed

399

between zebrafish sired by cryopreserved sperm is highly dependent of the

400

cryoprotectants composition used in the freezing medium (Fig 4A). However, sperm

401

cryopreserved with treatments containing 15% of DMF generated a reduction of the

402

incidence of severe skeletal malformations on the offspring in relation to fresh sperm

403

(Fig 4B). Sperm cryopreserved with Ctrl and EY treatments containing 12.5% of

404

DMF showed an increase in severe skeletal malformation when compared to fresh

405

sperm, however EY treatment with 12.5% of DMF had a low number of analyzed

406

individuals (14 fish). Severe skeletal malformations of zebrafish sired by

407

cryopreserved sperm, namely lordosis, scoliosis and kyphosis, were reduced when

408

non-permeating cryoprotectants were added to a freezing medium with 15% of

409

DMF. The BSA treatment resulted in very low survival and no skeletal analysis was

410

performed. The axial skeleton malformations in zebrafish were mainly focused on

411

the caudal and caudal fin vertebrae (Fig 5A-C). Sperm cryopreserved with 12.5%

412

DMF and Gly showed particularly high percentage of malformations in caudal

413

vertebrae where each individual showed multiple malformations in the same region

414

considering that there are more malformations then individuals (percentage above

415

100%). Offspring generated by cryopreserved sperm display predominantly a load of

416

2 anomalies on the axial skeleton, although not significant (Fig 5D and E). These

417

anomalies were located on the transition between caudal vertebrae and caudal fin

418

vertebrae (vertebrae 27-30) (Fig 6). In figure 6 are represented some of the most

419

common skeletal malformations observed. In this figure is represented a fusion in

420

precaudal vertebrae (Fig 6A), abnormal vertebral bodies with ectopic calcifications

421

(Fig 6B) and a fish with absent hypural connection to the urostyle (Fig 6C).

422

Additionally, in this figure is represented a fish with malformed secondary haemal

17

423

arch on vertebrae number 29 with demineralization of hypural (Fig 6D), an

424

individual with abnormal neural arches, with ectopic calcification on parhypural and

425

demineralization in hypurals (Fig 6E) and a fish with severe scoliosis (Fig 6F).

426

To explore the potential relationships between cryoprotectant composition and the

427

offspring skeletogenesis, the complete description of skeletal abnormalities was

428

applied to a decision tree through CART method (Fig 7). Considering if the fish

429

were malformed or displayed a normal skeletal development (dependent variable),

430

the severity of skeletal malformations was the factor that discriminate treatments the

431

most, followed by the incidence of abnormalities on the arches (Fig 7).

432

The decision tree allows to observe that the use of 15% of permeating cryoprotectant

433

(DMF) on cryopreserved treatments reduces the onset of deformed arches on the

434

offspring sired by cryopreserved sperm. Among the fish without severe skeletal

435

malformations but containing deformed arches, the treatments that show lower

436

number of deformed fish were obtained with an extender containing 15% of DMF

437

(node 10), which is observed on the left branch of the decision tree. The visualization

438

of the nodes on the right branches allow to observe that fish with no severe skeletal

439

malformations, no deformed arches, no fusions, no compressions (node 0, 2, 4, 7,

440

12) lead to the three optimal treatments regarding offspring skeletal development

441

(higher number of normal fish) namely 15% DMF Ctrl, 15% DMF EY, 15% DMF

442

Bici (node 14) (Fig 7). The use of non-permeating cryoprotectants was discriminated

443

through the incidence of fusions (node 7), and on a subsequent tree node, vertebral

444

compression (node 12) on the offspring where two non-permeating cryoprotectant

445

groups were formed. The group formed by fresh sperm, BSA and glycine with

446

12.5% of DMF show lower number of normal individuals (53.8%) and the group

18

447

formed by control, egg yolk and bicine treatments (72.9%) show significantly higher

448

number of normal individuals (P = 0.039).

449 450

Discussion

451

The ultimate objective of assisted reproduction techniques such as sperm

452

cryopreservation is not only to accomplish oocyte fertilization, but most importantly

453

to obtain viable and healthy offspring. Spermatozoa are more than carriers of

454

genomic information, they have a crucial role on the genetic control of the first

455

embryonic events after fertilization [18, 40, 45, 71, 74]. However, the spermatozoa

456

ability to repair DNA damage is absent [59, 67] and depend on oocyte DNA repair

457

machinery to perform its genomic repair, onto some extent [29]. Cryopreservation

458

can produce oxidative stress and increase sperm DNA damage [15, 25, 50]. In fact,

459

the fertilization with high DNA damaged spermatozoa is an important factor that

460

leads to abortion [19, 59]. Nevertheless, beyond spermatozoa lethal DNA damage,

461

there are sub-lethal damage induced by cryopreservation that affect progeny quality,

462

such as longer telomeres on offspring [60], abnormal juvenile weight and cortisol

463

response to stress [38], malformations at hatching [56, 79] and haploidy [56]. The

464

effects of the damage produced during sperm cryopreservation on offspring quality

465

and development are still poorly investigated. Consequently, with our work we

466

aimed to investigate the effect of different concentrations of the permeating

467

cryoprotectant with and without the addition of different non-permeating

468

cryoprotectants on sperm quality, and on the skeletal malformations of the offspring

469

sired by cryopreserved sperm.

470

Sperm cryopreservation methodologies must be adapted for each species (and cell

471

type) since cells response to the freezing process depend on the cell biophysical

19

472

characteristics, which are species specific and change in a nonlinear mode with the

473

freezing temperature throughout the cryopreservation process [22]. Data show total

474

post-thaw sperm motility and viability values within the previously reported in

475

zebrafish [25, 72, 77]. There are few reports of zebrafish hatching rates obtained

476

with cryopreserved sperm [25, 37]. Hatched larvae obtained by in vitro fertilization

477

with fresh sperm in our work yielded an average of 23%, whereas in Diogo et al.

478

[25] an average of 12% was reported. Consequently, the assisted reproduction

479

methods may impact negatively the hatching rates of zebrafish as in mammalian

480

species [62]. Our data show no significant differences in embryo survival 24 hpf and

481

hatching rates due to the high variability among samples. The variability observed

482

occurs even though males and samples are thoroughly selected according to

483

previously established thresholds of male age, sperm total motility and cell

484

concentration [25, 26]. Therefore, it is important to acknowledge that sperm

485

cryopreservation in this species is difficult, not only due to the low sperm volume

486

and low post-thaw sperm quality, but also due to the highly variable in vitro

487

fertilization success, both in fresh and cryopreserved sperm. This bottleneck is

488

particularly relevant while performing sperm cryopreservation of highly valuable or

489

vulnerable zebrafish lines, because it is challenging to obtain viable embryos with

490

post-thaw sperm for the restoration of the zebrafish lines.

491

Permeating cryoprotectants are among the most relevant players for cryopreservation

492

success, they permeate sperm plasma membrane and increase total intracellular

493

solute concentration [28, 50]. Consequently, water leaves the cells through osmotic

494

gradient, avoiding the formation of intracellular ice crystals, which are lethal to the

495

cell [54, 55]. The disadvantage of permeating cryoprotectants is their toxicity and

496

therefore, to accomplish a feasible cryopreservation protocol, a compromise between

20

497

low toxicity and complete cellular penetration must be attended [3, 28, 50, 55]. In

498

our work, the post-thaw total and progressive sperm movement as well as in vitro

499

fertilization parameters show a normal distribution, which represents the balance

500

between cryoprotectant toxicity and cellular protection against cold damage. Data

501

showed that low variations on permeating cryoprotectant concentrations impacts

502

post-thaw sperm quality. The freezing medium containing 5% of DMF was

503

deleterious to sperm in all quality parameters, especially in 24 hpf embryo survival

504

and hatching rates. This result suggests that 5% of DMF is not enough to protect

505

zebrafish spermatozoa from the cryopreservation process. Post-thaw sperm total

506

motility and membrane viability was improved by 12.5% and 15% of DMF. These

507

DMF concentrations produced hatching rates similar to fresh sperm and the highest

508

of the cryopreserved treatments. Therefore, these DMF concentrations were used to

509

study the interaction of permeating with non-permeating cryoprotectants.

510

Non-permeating cryoprotectants such as sugars and amino acids are able to establish

511

interactions with membrane lipidic bilayers [17], protecting the cells during the

512

freezing process and improving post-thaw results [15, 49]. In our work, the addition

513

of BSA and Gly to the freezing medium yielded lower progressive motility.

514

Permeating and non-permeating cryoprotectants in our study showed significant

515

interaction in total motility. This synergy between DMF concentration and non-

516

permeating cryoprotectants suggest that BSA and Gly show a reduction in total

517

motility with 15% DMF, while the treatments that yielded the best results (EY, Bici

518

as well as Ctrl) maintain high total motility with both DMF concentrations. These

519

combinations may protect the plasma membrane components responsible for the

520

triggering of zebrafish sperm motility. It is interesting to observe that the additives

521

used in the freezing media composition reduce the sperm velocities and linearity

21

522

when compared to the control. However, lower sperm velocity does not result in

523

lower 24 hpf embryo survival and hatching rates. Egg yolk yielded high post-thaw

524

sperm quality and hatching rates, which might be explained by its high viscosity that

525

protects the cell during cryopreservation [57] and the increase of the flagellar beating

526

frequency in viscoelastic fluids [47]. Viscosity stabilizes the fertilization

527

microenvironment, which is important in teleosts external fertilization [46],

528

particularly in species that yield low sperm volume such as Senegalense sole [23, 63]

529

and zebrafish. The main disadvantages of egg yolk are the difficult standardization

530

and high susceptibility to contamination [1].

531

Bicine is an amino acid [N,N-Bis(2-hydroxyethyl)glycine] with high buffer capacity

532

and recommended for biological research at low temperatures [35]. Bicine is

533

commonly used in freshwater species freezing media [14] and was recently used in a

534

zebrafish sperm cryopreservation protocol [53]. However, its isolated effect on post-

535

thaw sperm quality required deeper comprehension. The use of 15% of DMF

536

significantly reduced plasma membrane viability when compared to 12.5% of DMF.

537

However, freezing media containing 15% of DMF with EY or Bici showed a

538

reduction of skeletal malformations severity on the resulting offspring. The same

539

results were observed in Ctrl (15% of DMF).

540

Sub-optimal cryopreservation protocols are known to produce genetic and epigenetic

541

alterations with negative consequences on offspring biological performance and

542

phenotype, affecting thus their health and lifespan [60]. Traditionally, skeletal

543

malformations are associated to nutritional factors, however the perturbation of

544

genes responsible for the ossification are known to be responsible for abnormal

545

skeletogenesis [48]. Sperm cryopreservation in trout was associated to the alteration

546

of genes involved in the regulation of embryo early development, particularly

22

547

symmetry, embryonic body axes development (e.g. anterior–posterior, dorsal–ventral

548

and left-right axis), segmentation, gastrulation, organogenesis and tissues

549

differentiation [29], which are associated to skeletal development. Our results

550

indicate that the severe skeletal malformations of the offspring sired by

551

cryopreserved sperm provides relevant information on the effectiveness of the

552

cryopreservation protocol, that would be otherwise disregarded. The skeletal

553

malformations incidence on zebrafish sired by fresh sperm through in vitro

554

fertilization are within the normal range for this species in natural spawns [24, 51].

555

In our work, severe skeletal malformations that change fish external body shape such

556

as lordosis, scoliosis and kyphosis were significantly higher with 12.5% of DMF

557

when compared to 15% DMF. This fact suggests that 15% of DMF can protect the

558

cell against residual intracellular ice crystals formation, cold damage or cellular

559

stress that lead to genomic alternations and consequently to abnormal skeletal

560

development. Interestingly, the offspring sired by cryopreserved sperm with 15% of

561

DMF with EY, Gly or Bici showed lower malformations when compared to fresh

562

sperm. These data can result from a synergy between multiple factors. Although, the

563

experimental design was conceived to reduce the maternal derived effects, it is not

564

possible to exclude this factor, which can partially explain this result. Moreover,

565

zebrafish has continuous reproduction with low sperm quality in fresh sperm [25,

566

26]. A possible explanation for the reduction of severe skeletal malformations for the

567

previously mentioned cryopreserved treatments, is the elimination of labile

568

spermatozoa (overripe or immature) present in fresh sperm during cryopreservation.

569

Therefore, the most suitable freezing media used in cryopreservation would preserve

570

the cells with highest quality, resulting in offspring with lower incidence of severe

571

malformations (i.e. lordosis, kyphosis and scoliosis). The severe malformations in

23

572

zebrafish juveniles with 30 days post fertilization can give insights on the alterations

573

in the early development of embryo structures and the genes responsible for their

574

regulation. On early embryo, three embryonic layers are formed through extensive

575

cellular rearrangements namely ectoderm, mesoderm and endoderm [5]. Each one of

576

these embryonic layers will originate different body structures. Zebrafish vertebrae

577

derive from notochord (originated from ectoderm) [30] while arches derive from

578

somite cellular line (originated from mesoderm) [5]. Therefore, the skeletal

579

malformations observed in the offspring sired by cryopreserved sperm can be caused

580

by alterations in spermatozoa genes related to the paternal contribution for the

581

regulation of ectoderm and mesoderm. This fact can further affect the development

582

of notochord and arches, respectively, producing malformed larvae with lower

583

biological fitness. These aspects should be further investigated.

584

The regions most affected by skeletal anomalies in our study were caudal and caudal

585

fin vertebrae, which is a typical location for the development of malformations in

586

this species [4, 24, 51]. In our study cryopreservation did not change this

587

malformation pattern and there were no differences in the charge of skeletal

588

malformations (number of malformations per individual) when compared to larvae

589

sired by fresh sperm. Therefore, these two parameters are less informative in the

590

quality evaluation of zebrafish sired by cryopreserved sperm. For this purpose, the

591

analysis of severe malformations was a suitable larvae quality biomarker.

592

Treatments with 15% of DMF with or without egg yolk or bicine reduced the onset

593

of deformed arches, vertebral fusions and compressions on the offspring. Therefore,

594

sub-optimal freezing medium composition in cryopreservation may cause a

595

perturbation of the early embryo genome and structures relevant for ossification,

596

disturbing thus zebrafish normal skeletal development. The detailed analysis of

24

597

skeletal malformations generates complex data sets with inherent high biological

598

variability, being therefore difficult to analyze through traditional statistical methods.

599

Using a decision tree with detailed information of offspring skeletal anomalies sired

600

with cryopreserved and fresh sperm it was possible to predict that 15% DMF

601

reduced the number of deformed fish which validates the observations of figure 4.

602

This fact suggests that 15% of DMF was able to protect spermatozoa genes involved

603

on embryo somitogenesis. Our work evidences that the freezing media composition

604

that yields consistently improved sperm and offspring quality is 15% DMF with or

605

without Bici or EY. Ey have associated sanitary risks and standardization

606

difficulties, therefore we suggest that 15% DMF with 50 mM Bici is the most

607

adequate cryoprotectant composition. Moreover, our work evidences that decision

608

trees are useful resources to be explored on the selection of sperm cryopreservation

609

protocols, since they generate a straightforward statistical discrimination of the

610

treatments.

611

To the best of our knowledge, this is the first report on the skeletal malformations

612

description of the offspring sired by cryopreserved sperm with different freezing

613

media compositions in zebrafish. Our work shows that sub-lethal damage of

614

spermatozoa resulting from under-optimized cryopreservation protocols can increase

615

the incidence of skeletal malformations in zebrafish offspring. Therefore, offspring

616

skeletal development evaluation is a valuable tool for the selection of efficient

617

cryopreservation protocols.

618 619

Acknowledgments

620

Patricia Diogo acknowledges the financial support from the Portuguese Foundation

621

for Science and Technology (FCT) through the doctoral grant

25

622

SFRH/BD/97466/2013. This study received Portuguese national funds from FCT -

623

Foundation for Science and Technology through project UID/Multi/04326/2019 and

624

from the operational programmes CRESC Algarve 2020 and COMPETE 2020

625

through project EMBRC.PT ALG-01-0145-FEDER-022121.The authors

626

acknowledge the Light Microscopy Unit of CBMR-UAlg, especially the

627

collaboration of the facility staff Claudia Florindo and Inês Baião-Santos. The

628

authors acknowledge Maurícia Vinhas for flow cytometry support, Elisabete Matos

629

and Matthew Castaldi for manuscript revision.

630 631

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improves growth, reproductive performance and reduces skeletal anomalies

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Examination of larval malformations in African catfish Clarias gariepinus

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following fertilization with cryopreserved sperm, Aquaculture 247 (2005)

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developmental success, Reproduction 139 (2010) 989-997.

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Herráez, Altered gene transcription and telomere length in trout embryo and

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larvae obtained with DNA cryodamaged sperm, Theriogenology 76 (2011)

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1234–1245.

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Soler, P. Jiménez-Rabadán, M.R. Fernández-Santos, R. Bernabéu, J.J. Garde,

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Taking advantage of the use of supervised learning methods for

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characterization of sperm population structure related with freezability in the

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Iberian red deer, Theriogenology 77 (2012) 1661–1672.

816 817

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technologies, Mol. Reprod. Dev. (2019) 1–15.

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821

Solea senegalensis sperm cryopreservation: New insights on sperm quality,

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839

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857

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858

production with cryopreserved sperm from a live-bearing fish Xiphophorus

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maculatus and implications for female fecundity, in: Comp. Biochem.

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13 (2016) 144–151.

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35

867

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869

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870

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(Oncorhynchus mykiss) produced with cryopreserved sperm, Aquaculture

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873

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L. Zhang, S. Wang, W. Chen, B. Hu, S. Ullah, Q. Zhang, Y. Le, B. Chen, P.

874

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875

zebrafish (Danio rerio) spermatozoa., Pak. Vet. J. 34 (2014) 518–521.

876 877

Fig 1. Representation of experimental design used for in vitro fertilization performed

878

with one sperm pool. This schematical representation was repeated for 6

879

sperm pools (containing sperm from 10 males) for experiment 1, and 5 sperm

880

pools (containing sperm from 16 males) for experiment 2. Experiment 1

881

assessed the effect of different concentrations of permeating cryoprotectant

882

(DMF) in zebrafish cryopreserved (Cryo) sperm. Experiment 2 assessed the

883

effect of non-permeating cryoprotectants [(Ctrl, 10 mg/ml of BSA (BSA),

884

10% egg yolk (EY), 30 mM glycine (Gly) and 50 mM of bicine (Bici)] with

885

12.5 and 15% DMF. Independent clutches of oocytes from different females

886

were fertilized with each sperm pool, with each experimental condition (fresh

887

and cryopreserved). For each treatment, each sperm pool was used to fertilize

888

2-5 clutches of oocytes (approximately 100-200).

889 890 891

Fig 2. Effect of different concentrations of permeating cryoprotectant (DMF) on zebrafish sperm (n = 6 pools containing sperm of 10 males) A) total motility

36

892

(%), B) progressive motility (%), C) curvilinear velocity (µm/s), D) straight-

893

line velocity (µm/s), E) linearity (%) and F) viability of the plasma membrane

894

(%). For the same pools of sperm, the success of in vitro fertilizations

895

performed with fresh (n = 4) and cryopreserved sperm with 5% (n = 5), 7.5%

896

(n = 4), 10% (n = 5), 12.5% (n = 5) and 15% (n = 4) was evaluated through

897

G) embryo survival 24 h post fertilization (%) and H) hatching rate at 72 h

898

post fertilization (%). The values plotted in white (fresh sperm) and black

899

(cryopreserved sperm) bars represent Mean ± SD. Different letters on the bars

900

indicate significant differences (one-way ANOVA, post hoc SNK P < 0.05).

901 902

Fig 3. Zebrafish sperm (n = 5 pools containing sperm of 16 males) either fresh

903

(white bars) or cryopreserved with 12.5% (black bars) and 15% (grey bars) of

904

DMF without non-permeating cryoprotectant (Ctrl) and with 10 mg/ml of

905

BSA (BSA), 10% egg yolk (EY), 30 mM glycine (Gly) and 50 mM of bicine

906

(Bici). Sperm quality was evaluated according to A) total motility (%), B)

907

progressive motility (%), C) curvilinear velocity (µm/s), D) straight line

908

velocity (µm/s), E) linearity (%) and F) viability of the plasma membrane

909

(%). For the same sperm pools in vitro fertilizations were performed and their

910

success was measured through G) embryo survival 24 h post fertilization (%)

911

and H) hatching rate at 72 h post fertilization (%). Values plotted represent

912

Mean ± SD, asterisk indicate significant differences between fresh and

913

cryopreserved sperm (independent samples t-test, P < 0.05). Uppercase letters

914

represent significant differences between permeating cryoprotectant

915

concentration and lowercase letters significant differences between non-

916

permeating cryoprotectants (two-way ANOVA, post hoc SNK P < 0.05).

37

917 918

Fig 4. Offspring axial skeleton malformations (30 dpf) analysis through alcian blue

919

alizarin red staining in terms of A) malformed fish (%), B) severe skeletal

920

malformations (%). White bars represent fresh sperm (43 fish resulting from

921

3 sperm pools). Black bars represent zebrafish that resulted from in vitro

922

fertilization with cryopreserved sperm with 12.5% of DMF without non-

923

permeating cryoprotectant (Ctrl, 233 fish that resulted from 8 sperm pools)

924

and with 10 mg/ml of BSA (BSA, 37 fish that resulted from of 2 sperm

925

pools), 10% egg yolk (EY, 14 fish that resulted from of 1 sperm pool), 30

926

mM glycine (Gly, 10 fish that resulted from 2 sperm pools) and 50 mM of

927

bicine (Bici, 90 fish that resulted from 4 sperm pools). Grey bars represent

928

zebrafish that resulted from in vitro fertilization with cryopreserved sperm

929

with 15% of DMF without non-permeating cryoprotectant (Ctrl, 59 fish that

930

resulted from 3 sperm pools) and with 10% egg yolk (EY, 168 fish that

931

resulted from of 7 sperm pools), 30 mM glycine (Gly, 37 fish that resulted

932

from 2 sperm pools) and 50 mM of bicine (Bici, 73 fish that resulted from 3

933

sperm pools). Values plotted represent Mean ± SD, different letters represent

934

significant differences between treatments, asterisk represent significant

935

differences in relation to fresh sperm (Chi-square P < 0.05).

936 937

Fig 5. Offspring axial skeleton malformations (30 dpf) analysis through alcian blue

938

alizarin red staining. A) representation of zebrafish axial skeleton (adapted

939

from Bird and Mabee 2003). Upper figures represent the distribution of

940

malformations through zebrafish skeleton of the offspring generated from

941

sperm cryopreserved with B) 12.5% of DMF and C) 15% of DMF. Lower

38

942

figures represent the charge of malformations (number of malformations per

943

individual) on the offspring generated from sperm cryopreserved with D)

944

12.5% of DMF and E) 15% of DMF. For each permeating cryoprotectant

945

concentration was tested a control without non-permeating cryoprotectant

946

(Ctrl, 233 fish that resulted from 8 sperm pools) and the addition of 10 mg/ml

947

of BSA (BSA, 37 fish that resulted from of 2 sperm pools), 10% egg yolk

948

(EY, 14 fish that resulted from of 1 sperm pool), 30 mM glycine (Gly, 10 fish

949

that resulted from 2 sperm pools) and 50 mM of bicine (Bici, 90 fish that

950

resulted from 4 sperm pools). Values plotted represent Mean ± SD and

951

different shades of grey represent zebrafish skeleton location (A and B) or

952

number of anomalies (C and D).

953 954

Fig 6. Representation of the most abundant axial skeleton malformations (30 dpf) of

955

zebrafish sired by cryopreserved sperm A) Fusion in precaudal vertebra

956

associated to compressive forces (black arrow), B) enlarge vertebral bodies

957

(white arrows) with ectopic calcifications (black arrows), C) absence of

958

hypural 1 connection to the urostyle (black arrow), D) secondary haemal arch

959

on vertebrae No. 29 (white arrow); demineralized hypural 1 (black arrow);

960

broken neural arch on urostyle (asterisk), E) abnormal neural arches (white

961

arrows); ectopic calcification on parhypural (black arrow); demineralized

962

regions in hypurals 1 and 2 (black arrows); broken neural arch on urostyle

963

(asterisk) F) Severe scoliosis associated to compressive forces (white arrow).

964 965 966

Fig 7. Decision tree obtained through CART method to predict the effect of the extender composition used in zebrafish cryopreservation in offspring skeletal

39

967

development. The skeletal malformations analysis details the occurrence of

968

severe skeletal anomalies: scoliosis, kyphosis, lordosis, compressions,

969

fusions, opened arches, deformed arches, deformed centra (vertebrae) for the

970

offspring generated by fresh and cryopreserved sperm. The zebrafish

971

analyzed were obtained from sperm cryopreserved with 12.5% and 15% of

972

DMF without non-permeating cryoprotectant (Ctrl, 233 fish that resulted

973

from 8 sperm pools) or with the addition of 10 mg/ml of BSA (BSA, 37 fish

974

that resulted from of 2 sperm pools), 10% egg yolk (EY, 14 fish that resulted

975

from of 1 sperm pool), 30 mM glycine (Gly, 10 fish that resulted from 2

976

sperm pools) and 50 mM of bicine (Bici, 90 fish that resulted from 4 sperm

977

pools). Statistical significance is represented in each tree node, when the tree

978

ramification stops no significant differences are observed within the group.

979

Each node is divided into a group with significantly higher presence of the

980

prementioned characteristic (e.g. severity) referred as Yes or significantly

981

lower presence of individuals with the same characteristic referred as No.

982

When the tree does not grow from a terminal or a characteristic is not

983

mentioned, means that there are no statistical differences among the analyzed

984

zebrafish. The treatments resulting in animals with healthier skeletogenesis

985

are located on the right branches following all the nodes, with no

986

deformations, where the right terminal shows the treatments characterized

987

with more normal individuals (node 14).

988 989

40

TABLE 1– Post-thaw zebrafish sperm quality analysis related to the effect of permeating cryoprotectant concentration, non-permeating cryoprotectants and their interactions in post-thaw zebrafish sperm. Two-way ANOVA (p value < 0.05) TM PM VCL VSL LIN Viability Embryo survival Hatching rate

Permeating

Non-permeating

Permeating*non-permeating

0.010* 0.284 0.114 0.082 0.076 0.050 0.925 0.406

0.003* 0.032* 0.023* 0.008* 0.005* 0.013* 0.105 0.289

0.047* 0.230 0.118 0.085 0.062 0.145 0.367 0.723

Significant differences (two-way ANOVA (SNK, p < 0.05)) are represented with asterisk.

Malformed or normal fish Node 0 Category % n Malformed 57.7 472 42.3 346 Normal Total 100.0 818

Malformed* Normal *fish exhibiting at least one skeletal anomaly

Severity Adjusted p-value=0.039

Yes

No

Node 1 Category % n 0.0 Malformed 0 100.0 45 Normal Total 5.5 45

Node 2 Category % n Malformed 61.1 472 38.9 301 Normal Total 94.5 773 Deformed arches Adjusted p-value=0.029

Yes

No

Node 3 Category % n Malformed 16.4 9 83.6 46 Normal Total 6.7 55

Node 4 Category % n Malformed 64.5 463 35.5 255 Normal Total 87.8 718

Treatment Adjusted p-value=0.003

Fusions Adjusted p-value=0.025

Fresh;12.5% DMF 12.5% ; 12.5% ; 12.5% ; 12.5% ; 15% Gly sperm Ctrl Bici EY Ctrl BSA Node 5 Category % Malformed 36.8 63.2 Normal Total

2.3

n 7 12 19

Yes

No

Node 6 Category % n 5.6 Malformed 2 94.4 34 Normal Total 4.4 36

Node 7 Category % n Malformed 66.9 461 33.1 228 Normal Total 84.2 689

Cryoprotectant Adjusted p-value=0.000

Compressions Adjusted p-value=0.018

12.5%

15%

Node 9 Category % n 0.0 Malformed 0 100.0 17 Normal Total 100.0 17

Node 10 Category % n Malformed 10.5 2 89.5 17 Normal Total 2.3 19

Yes

Node 8 Category % n 6.9 Malformed 2 93.1 27 Normal Total 3.5 29

No

Node 11 Category % n 0.0 Malformed 0 100.0 16 Normal Total 2.0 16

Node 12 Category % n Malformed 68.5 461 31.5 212 Normal Total 82.3 673 Non-permeating cryoprotectant Adjusted p-value=0.039

Fresh ; 12.5% ; 12.5% Gly sperm BSA Node 13 Category % Malformed 53.8 46.2 Normal Total

n 84 72 19.1 156

15% ; 15% ; 15% Ctrl EY Bici Node 14 Category % n Malformed 72.9 377 27.1 140 Normal Total 63.2 517

Highlights of Diogo et al manuscript: “Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis“

-

Cryoprotectant composition of extender affect skeletal development of the offspring

-

15% dimethylformamide in extender decrease offspring severe skeletal malformations

-

Cryopreservation with 15% of DMF with bicine or egg yolk improve zebrafish post-thaw sperm quality

-

Bicine or egg yolk in extender reduce offspring vertebral fusions and compressions