A laboratory guide to in vitro fertilization of oceanic squids

Abstract

In vitro fertilization of oceanic squid is a necessary step to develop their larval culture and creates new opportunities to study and understand cephalopod development, taxonomy and ecology. The techniques described here in the form of a laboratory guide represent an attempt to refine and standardize the general methodology by indicating suitable laboratory materials, sources and preservation of gametes, and methods for fertilization and egg incubation. Twelve oceanic squid species have been fertilized in vitro to date; we outline a generalized experimental protocol and suggest that the reader consider particular species-specific modifications. Inadequate egg chorion expansion and premature hatching are identified as major challenges for in vitro fertilization. Recommendations for future research include studies on optimal gamete concentration, gamete preservation and determination of the functions of female oviducal and nidamental glands. The greatest obstacles to improving fertilization success in squids are the lack of standard methodologies and the paucity of information on both endogenous and exogenous factors controlling the fertilization process. This review is a first step toward overcoming these challenges.

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Review
A laboratory guide to in vitro fertilization of oceanic squids
Roger Villanueva
a,
⁎, Danna J. Staaf
b
, Juan Argüelles
c
, Anna Bozzano
a
, Susana Camarillo-Coop
d
,
Chingis M. Nigmatullin
e
, Giuliano Petroni
a
, Daniel Quintana
a
, Mitsuo Sakai
f
, Yasunori Sakurai
g
,
César A. Salinas-Zavala
d
, Roxana De Silva-Dávila
h
, Ricardo Tafur
c
, Carmen Yamashiro
c
, Erica A.G. Vidal
i
a
Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, E-08003 Barcelona, Spain
b
Hopkins Marine Station of Stanford University, Oceanview Blvd, Pacific Grove, California 93950, USA
c
Instituto del Mar del Peru (IMARPE), Esquina Gamarra y General Valle s/n, Chucuito, Callao, Peru
d
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo # 195 Col. Playa Palo de Santa Rita, C.P. 23090 La Paz, B.C.S. Mexico
e
Laboratory of Commercial Invertebrates, Atlantic Research Institute of Fisheries and Oceanography (AtlantNIRO), Dm. Donskoy st. 5, Kaliningrad 236000, Russia
f
National Research Institute of Far Seas Fisheries, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa-ku, Yokohama-shi, 236-8648 Japan
g
Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido, 041-8611, Japan
h
Centro Interdisciplinario de Ciencias Marinas (CICIMAR-IPN), Departamento de Plancton y Ecología Marina, Av. IPN s/n, Col Playa Palo de Santa Rita, La Paz, B.C.S., Mexico
i
Centro de Estudos do Mar, Universidade Federal do Paraná (UFPR), Cx. Postal 50.002, Pontal do Paraná, PR. 83.255-000 Brazil
abstractarticle info
Article history:
Received 5 August 2011
Received in revised form 23 February 2012
Accepted 23 February 2012
Available online 3 March 2012
Keywords:
Cephalopoda
Teuthida
Gametes
Embryonic development
Paralarvae
In vitro fertilization of oceanic squid is a necessary step to develop their larval culture and creates new oppor-
tunities to study and understand cephalopod development, taxonomy and ecology. The techniques described
here in the form of a laboratory guide represent an attempt to refine and standardize the general methodol-
ogy by indicating suitable laboratory materials, sources and preservation of gametes, and methods for
fertilization and egg incubation. Twelve oceanic squid species have been fertilized in vitro to date; we outline
a generalized experimental protocol and suggest that the reader consider particular species-specific modifi-
cations. Inadequate egg chorion expansion and premature hatching are identified as major challenges for in
vitro fertilization. Recommendations for future research include studies on optimal gamete concentration,
gamete preservation and determination of the functions of female oviducal and nidamental glands. The
greatest obstacles to improving fertilization success in squids are the lack of standard methodologies and
the paucity of information on both endogenous and exogenous factors controlling the fertilization process.
This review is a first step toward overcoming these challenges.
© 2012 Elsevier B.V. All rights reserved.
Contents
1. Introduction .............................................................. 126
2. Laboratory guide to in vitro fertilization of oceanic squids ........................................ 127
2.1. Species-specific considerations .................................................. 127
2.2. Sources and preservation of gametes ............................................... 127
2.2.1. Oocytes ........................................................ 127
2.2.2. Spermatozoa ...................................................... 127
2.2.3. Age and preservation of gametes ............................................ 129
2.3. Laboratory materials ...................................................... 129
2.3.1. Laboratory tools .................................................... 129
2.3.2. Seawater ........................................................ 129
2.3.3. Oviducal gland jelly and other substances to obtain chorion expansion ........................... 129
2.3.4. Anaesthetic ...................................................... 129
2.4. Step-by-step recipe for in vitro fertilization and egg incubation .................................. 129
2.4.1. Activation of spermatozoa ............................................... 129
2.4.2. Collection of oocytes .................................................. 130
Aquaculture 342–343 (2012) 125–133
⁎Corresponding author. Tel.: +34 932 309 500; fax: +34 932 309 555.
E-mail address: roger@icm.csic.es (R. Villanueva).
0044-8486/$ –see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.02.025
Contents lists available at SciVerse ScienceDirect
Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Author's personal copy
2.4.3. Fertilization ...................................................... 130
2.4.4. Egg incubation ..................................................... 130
3. Future research ............................................................ 130
4. Conclusions .............................................................. 131
Acknowledgments .............................................................. 131
References ................................................................. 131
1. Introduction
Oceanic squids (Teuthida: Oegopsida) are one of the most diverse
groups of cephalopods, with more than 240 species described, occu-
pying key trophic roles as predators in the open ocean ecosystem
(Clarke, 1996; Jereb and Roper, 2010). Some of these species undergo
high fishing pressure and their catches represent half of the total
cephalopod world captures (Boyle and Rodhouse, 2005; FAO, 2010).
But despite accumulated knowledge on the biology and ecology of ju-
venile, subadult and adult forms, information on eggs and paralarvae
is still very limited and, for most species, does not exist at all.
This is probably due to their oceanic life and spawning mode.
Neritic loliginid squid species attach dense egg masses to hard sur-
faces in shallow waters which are easily accessible to humans; by
contrast, most oceanic squids produce (or are suspected to produce)
ellipsoid translucent balloons from one to four meters in diameter
containing from tens to hundreds of thousands of eggs. These egg
masses are released in the open ocean, making them difficult for re-
searchers to detect and access (see among others Bower and
Sakurai, 1996; O'Shea et al., 2004; Roberts et al., 2011; Staaf et al.,
2008; Young et al., 1985a and Table 1). Unusual cases of species
spawning free eggs (Young et al., 1985b) or cylindrical egg masses
(Guerra et al., 2002; Miyahara et al., 2006b) or even brooding egg
masses in the female's arms (Bjorke et al., 1997; Seibel et al., 2000,
2005) have also been documented.
In view of the very low probability of collecting wild egg masses
and the difficulties involved in obtaining them from spawning of cap-
tive broodstock maintained in aquaria (Bower and Sakurai, 1996;
O'Dor and Balch, 1985; Staaf et al., 2008), in vitro fertilization tech-
niques have provided an alternative method for obtaining valuable
information on the early development of oceanic squids and are a
necessary step to develop their larval culture. Eggs of twelve oceanic
squid species (Table 1) have been fertilized artificially in the laborato-
ry, enabling the description of embryo morphology, determination of
the duration of embryonic life under different conditions and use of
hatchling specimens for paralarval identification and taxonomy
(Sakai et al., 1998; Sakurai et al., 1995; Watanabe et al., 1996; Yatsu
et al., 1999). These techniques have also facilitated the investigation
of the development of internal organs of embryos and hatchlings
(Shigeno et al., 2001a,b), somatic growth using statoliths (Balch et
al., 1988; Sakai et al., 1998, 2004; Yatsu et al., 1999), and swimming
behaviour of paralarvae (Staaf et al., 2008). These laboratory studies
reveal the temperature ranges in which normal embryogenesis is
possible and help to delimit potential spawning areas in the wild
Table 1
Studies on the eggs, egg masses and embryonic development of oegopsid oceanic squids. Wild and aquaria spawned egg masses as well as laboratory in vitro fertilization experi-
ments are indicated.
Species Fertilization type Geographic area Reference
Abralia trigonura Wild eggs North PacificBigelow, 1992
Abralia sp. In vitro North PacificArnold and O' Dor, 1990
Abraliopsis sp. In vitro North PacificArnold and O' Dor, 1990
Brachioteuthis sp. Wild eggs North PacificYoung et al., 1985b
Dosidicus gigas In vitro SE PacificYatsu et al., 1999
Wild egg mass, spawning in
aquaria and in vitro
NE PacificStaaf et al., 2008, 2011
Enoploteuthinae Wild eggs North PacificOkiyama and Kasahara, 1975; Young and Harman, 1985
Gonatus fabricii Wild egg mass NE Atlantic Bjorke et al., 1997
Gonatus onyx Wild egg mass NE PacificSeibel et al., 2000, 2005
Illex argentinus In vitro SW Atlantic Sakai and Brunetti, 1997; Sakai et al., 1998, 2004, 2011
Illex coindetii Wild egg mass Mediterranean Naef, 1928
Spawning in aquaria Mediterranean Boletzky et al., 1973
In vitro Mediterranean Villanueva et al., 2011
Illex illecebrosus Spawning in aquaria NW Atlantic Balch et al., 1985; Durward et al., 1980; O'Dor and Balch, 1985
Spawning in aquaria and in vitro NW Atlantic O'Dor et al., 1982
Lycoteuthidae Wild egg mass SW Indian Roberts et al., 2011
Nototodarus gouldi Wild egg mass SW PacificO'Shea et al., 2004
Ommastrephes bartramii In vitro North PacificSakurai et al., 1995
Sthenoteuthis oualaniensis Spawning in aquaria Arabian Sea Chesalin and Giragosov, 1993
In vitro North PacificSakurai et al., 1995
Sthenoteuthis pteropus Wild egg mass Eastern Atlantic Laptikhovsky and Murzov, 1990
Sthenoteuthis sp. In vitro North PacificArnold and O' Dor, 1990
Thysanoteuthis rhombus Wild egg mass East Atlantic and Mediterranean Guerra et al., 2002; Sanzo, 1929
Wild egg mass East PacificSabirov et al., 1987
Wild egg mass West PacificBillings et al., 2000
Wild egg mass NW PacificMisaki and Okutani, 1976; Miyahara et al., 2006a,b;
Suzuki et al., 1979; Watanabe et al., 1998
In vitro North PacificArnold and O' Dor, 1990
Todarodes pacificus In vitro NW PacificHayashi, 1960; Ikeda and Sakurai, 2004; Ikeda et al., 1993; Ikeda
and Shimazaki, 1995; Sakurai et al., 1995, 1996; Shigeno et al., 2001a,b;
Soeda, 1952, 1954, 1956; Watanabe et al., 1996;
Spawning in aquaria NW PacificBower and Sakurai, 1996; Hamabe, 1962, 1963
Todaropsis eblanae In vitro Mediterranean R. Villanueva, unpublished
Watasenia scintillans Spawning in aquaria NW PacificHayashi, 1995
126 R. Villanueva et al. / Aquaculture 342–343 (2012) 125–133

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(O'Dor et al., 1982; Sakurai et al., 1996; Staaf et al., 2011). Ultimately,
artificial fertilization may provide material for larval rearing experi-
ments to study the poorly understood physiology and ecology of
young oceanic squid. In vitro fertilization of two shallow water loligi-
nid squids, Doryteuthis pealeii,(Crawford, 1985; 2000, 2001, 2002,
2003; Kao, 1985; Klein and Jaffe, 1984; Wadeson and Crawford,
2003) and Heterololigo bleekeri (Iwata et al., 2011) has facilitated re-
search like that described above. However, due to the aforementioned
ease of obtaining naturally spawned loliginid eggs, in vitro work on
loliginids has not been widespread.
The 5th International Symposium on Pacificsquid,heldduringOc-
tober 2010 in La Paz, Mexico, included a workshop entitled “Consider-
ations for in vitro embryonic development in Dosidicus gigas: theory
and practice.”As a conclusion of the workshop, participants compiled
their published and unpublished methodologies into a user-friendly
“cooking recipe”to be used in future research. The following sections
describe these methods and protocols and highlight areas that need
further investigation. Our objective is to establish a laboratory protocol
that ensures maximal success of in vitro fertilization of oceanic squid,
while identifying the main gaps in knowledge that are inhibiting fur-
ther success. Most knowledge of in vitro fertilization of squid comes
from a few species of the family Ommastrephidae; the reader should
consider that these techniques could be subjected to particular modifi-
cations when other families are studied.
2. Laboratory guide to in vitro fertilization of oceanic squids
2.1. Species-specific considerations
Before planning any in vitro fertilization experiments, a minimal
knowledge of the biology of the selected species and the oceano-
graphic conditions within its geographic range is necessary. Knowl-
edge of the species' maturity season(s) and spawning grounds helps
to identify the best time to collect mature individuals and carry out
experiments. Furthermore, it is helpful to know a well-defined sexual
maturity scale and maturity characteristics of the species, in order to
identify and select the best functionally mature individuals as sources
of gametes. The location of sperm storage in copulated females also
varies between species (see below). Reproductive anatomy in
Ommastrephid squids are showed in Fig. 1.
Oocyte size and egg incubation temperature influence the relative
duration of embryonic development in different species of cephalopods
(Boletzky, 1994; Laptikhovsky, 1999), and may determine the duration
of designed experiments. For example, oceanic squid species with rela-
tively small oocytes, such as Todarodes pacificus, whose oocyte length is
0.83 mm (Watanabe et al., 1996), need 2 days to hatch when incubated
at 26 °C and 8 days to hatch at 14 °C (Sakurai et al., 1996). In contrast, a
deep-sea species such as Gonatus onyx, with relatively large eggs of
2–3 mm in length, may need up to 9 months to develop at 3 °C, the
temperature at which spawning females were collected (Seibel et al.,
2000). Information about the oceanographic characteristics of the spe-
cies range and the depth ranges of known (or suspected) spawning are
important parameters to be taken into account when setting up labora-
tory temperature ranges. Suitable salinity should also be chosen based
on the species range.
2.2. Sources and preservation of gametes
Using as many adult females and males as possible at the same
time is recommended in order to select the most suitable gametes,
as there can be significant individual variability in gamete viability.
The female and male sources of gametes used in each fertilization
should always be recorded. A few hours after fertilization (see
below), the fertilization rates of the different progenitors can be
checked in order to select the most suitable group of embryos to con-
tinue incubating. The sequence of laboratory steps for the gamete
collection, in vitro fertilization and egg incubation of oceanic squid
are showed in Fig. 2.
2.2.1. Oocytes
In the present paper we use the term oocyte to refer the unferti-
lized female gametocyte. Oocytes are stored inside the oviducts of a
mature female, ready to be spawned. The term egg is used here to de-
note the zygote and resulting embryo obtained after fertilization.
Oocytes can be obtained from both oviducts of dissected mature
females; no difference in oocyte maturity between right and left
oviducts has been reported to date for oceanic squid.
2.2.2. Spermatozoa
Sperm can be collected from two sources: mature males and cop-
ulated females. Males of oceanic squid produce spermatophores in
the spermatophoric glands; the spermatophores are then
ov
ov
od
od
og
ng
spg
og
ng
ng
od
AB
ts ts
vd
vd
sg
sg
to
ss
sp
CD
Fig. 1. Reproductive anatomy of Ommastrephid squids. A, mature female Dosidicus
gigas; B, mature female Illex coindetii (note that nidamental glands have been displaced
laterally to shown the spermatangia); C, mature male Dosidicus gigas; D, mature male
Illex argentinus. Abbreviations: ng, nidamental gland; od, oviduct; og, oviducal gland;
ov, ovary; sg, spermatophoric gland; sp, spermatophores (released); spg, spermatan-
gia; ss, spermatophoric sac; to, terminal organ (passes under the gill); ts, testis;
vd, vas deferens.
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accumulated in the spermatophoric sac (Needhman's sac)
(Nigmatullin et al., 2003). During mating, the spermatophores under-
go the so-called spermatophoric reaction, a complex process of evag-
ination of the ejaculatory apparatus of the spermatophore that leads
to the extrusion and attachment of the spermatangium (i.e., everted
spermatophore containing the sperm mass) on different areas of the
female body (Marian, 2011, 2012; Nesis, 1995). Depending on the
species, the spermatangia (everted spermatophores) of copulated fer-
males can be found on: 1) the external body surfaces of the females as
modified seminal receptacles on the buccal membrane and outer lip
(Ikeda and Sakurai, 2004; Ikeda et al., 1993); 2) implanted in the an-
terior dorsal and ventral rugose, semi-gelatinous mantle tissue
Oocytes
Cut the oviduct
membrane and extract
oocytes from deep inside
with a sterile spoon, place
into sterile dish and cover
Cut the entire oviduct, drip
oocytes out into a sterile
dish and cover
Cut spermatophores or spermatangia in small
portions on a sterile dish until a homogenized mass
is obtained
Sperm
Place spermatangia on 1mm mesh and flush with
FSW to remove organic debris
Transfer the chopped spermatophores or
spermatangia to a glass container
Large oviduct species (Dosidicus)Small oviduct species (Illex)
100-200 oocytes per dish
Fertilization
Egg incubation
Incubation time: 2–3 hr
Replace FSW+A once a
day (or as many times as
possible if antibiotics are
not used) until hatching
Add oviducal gland jelly + antibiotics
to cover all the eggs
Gently stir
Transfer dishes to the incubator
Transfer fertilized
eggs to new sterile
dishes with FSW+A
Spermatophores
Spermatangia
Illex Dosidicus
Egg density:
• without antibiotics: 10–15 eggs dish-1 of 5 cm Ø
• with antibiotics: 60 eggs dish-1 of 6 cm Ø ( 5 eggs ml-1)
Fertilized eggs
are spherical
Unfertilized oocytes
are ovoid
Sperm suspension
Check spermatozoa mobility
under microscope
Drop sperm suspension with sterile
pipette to cover the oocytes within 25 min
of spermatozoa activation
Carefully mix with tip of pipette
Gamete contact time: 20-30 min
Add FSW+A for spermatozoa activation
Agitate for 3-5 min and filter with 150 µm
mesh to remove debris
Fig. 2. Schematic sequence of laboratory steps for the gamete collection, in vitro fertilization and egg incubation of oceanic squid. FSW, filtered seawater; FSW +A, filtered seawater
with antibiotics.
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(Hoving et al., 2008); 3) inserted in unmodified tissues on the skin of
the arms, head and mantle (Guerra et al., 2004; Hoving and
Laptikhovsky, 2007; Hoving et al., 2004, 2012; Norman and Lu,
1997); 4) attached internally on seminal receptacles situated on the
nuchal cartilage (Hoving et al., 2007); 5) implanted into the mantle
muscle layers (Hoving et al., 2010; Nesis et al., 1998), or 6) attached
to the inner surface of the mantle near the bases of the gills
(Brunetti, 1990; Sakai et al., 1998).
Ikeda et al. (1993) studied the fertilizing capacity of spermatozoa
from different parts of the mature male reproductive system and
from copulated females of T. pacificus, and concluded that spermato-
phores from the male's spermatophoric sac and spermatangia from
copulated females have similar fertilization rates. These authors
found that sperm from the male's vas deferens had less fertilizing ca-
pacity; however, Sakai et al. (2011) found high fertilization rates from
sperm collected from the male vas deferens in Illex argentinus.
2.2.3. Age and preservation of gametes
Most in vitro experiments to date have been carried out on board
oceanographic vessels immediately after capture of adult squid
(Arnold and O' Dor, 1990; Sakai and Brunetti, 1997; Sakai et al.,
1998; Sakurai et al., 1995; Staaf et al., 2008; Yatsu et al., 1999)or
from squid maintained in aquarium (Ikeda et al., 1993; Sakurai et
al., 1996; Watanabe et al., 1996) and in both cases showed high fertil-
ization rates. Fertilizations conducted 4–5 h after squid capture and
death have also demonstrated good fertilization rates, when whole
individuals are maintained on ice (mean: 67%, range: 28–94%)
(Villanueva et al., 2011). Whole individuals stored at 0 °C also main-
tain a high fertilization capacity (>72% within 24 h) (Sakai et al.,
2011). Use of gametes from recently dead squids is recommended;
however, gametes from oviducts and spermatophoric sacs placed in
sealed plastic bags and preserved at 0–4 °C can serve as relatively
good experimental material for up to two days (Staaf et al., 2011).
This time period may allow transport of gametes from the sea to a
land laboratory with better working facilities, or exchange of material
between laboratories.
Even greater longevity has been observed in sperm, but not in oo-
cytes. Spermatangia of the oceanic squid Dosidicus gigas stored in fil-
tered seawater at 12 °C showed spermatozoa motility for up to 5 days
(Huffard et al., 2007). Intact spermatophores of the cuttlefish Sepia
apama placed in plastic tubes with filtered seawater and refrigerated
at 4 °C showed spermatozoa motility after two months (Naud and
Havenhand, 2006). Long term sperm refrigeration has been not
been studied in squids, but similar characteristics may be suspected.
Preliminary results on the cryopreservation of Illex coindetii sperm
consisted of freezing the whole spermatophore in liquid nitrogen,
using dimethyl sulfoxide as cryoprotectant agent (Robles et al.,
submitted for publication). These spermatophores stored at 4 °C in
filtered seawater during two days after squid capture and then freez-
ing in liquid nitrogen allows the recovery of only a low percentage
(10%) of viable and motile spermatozoa.
2.3. Laboratory materials
2.3.1. Laboratory tools
Microbial infection is a major problem to avoid during fertilization
and egg incubation experiments, so the use of sterile material is es-
sential. Laboratory working surfaces, needles, forceps and scissors
should be cleaned with ethanol prior to use. Gloves and masks are
recommended. To date, all successful egg incubations have been per-
formed at a small scale using Petri dishes. Small or medium-sized
sterile dishes are recommended (50 to 90 mm diameter). If a dish is
infected, all the eggs inside will die within a few hours; multiple
small dishes therefore result in higher survival rates than one large
dish. Incubators for dishes are useful to maintain a stable temperature
as well as a clean incubation environment.
2.3.2. Seawater
Use of 0.2 μmfiltered seawater (FSW) is recommended. FSW can be
stored before use in autoclaved glass bottles in darkness at 4 °C. Recent-
ly, the addition of antibiotics (25 mg l
−1
each of ampicillin and strepto-
mycin) to FSW has provided high embryonic survival rates (Staaf et al.,
2008; Villanueva et al., 2011). However, previous studies had also
obtained high survival without antibiotics by changing FSW as frequent-
ly as possible to reduce microbial growth. When using antibiotics, the
mix of FSW+ antibiotic should be made new every day before use to
avoid antibiotic degradation. All FSW + antibiotic used during the
experiment should be collected for suitable waste treatment.
2.3.3. Oviducal gland jelly and other substances to obtain chorion
expansion
Obtaining expansion of the primary egg envelope, or chorion, is a
major challenge of in vitro fertilization. This requires the presence of
oviducal gland jelly in the incubation medium, as demonstrated by
Ikeda et al. (1993). Fresh, frozen or freeze-dried oviducal gland can
be used. To prepare fresh and frozen gland, pieces of tissue are
chopped with seawater and stirred just before fertilization. Freeze-
dried material should be shaken or blended to obtain fine powder,
which can be stored frozen until use. Freeze-dried oviducal gland is
highly recommended because the freeze-drying process reduces
pathogens, the powder is easy to transport and store, and dry weight
of the powder can be used to determine the exact quantities to add to
seawater. Good embryo survival and chorion expansion was observed
with concentrations of 1 g l
−1
of oviducal gland powder in seawater
(Sakurai et al., 1995; Villanueva et al., 2011). Oviducal gland powder
produces a mucus-like jelly solution after being stirred with FSW.
This solution often contains some organic debris, which should be re-
moved by filtering with a 100 μm mesh before the solution is added
to the eggs. The active component or characteristic of the oviducal
gland that helps chorion expansion remains unknown, but appears
to function across species. Within ommastrephids, oviducal gland
powder from one species can be used successfully with the eggs of
another species (Sakurai et al., 1995; Yatsu et al., 1999).
As an alternative to the use of oviducal gland jelly, in vitro fertiliza-
tion of Doryteuthis p ealeii was obtained with suitable chorion expansion
and hatching by using plastic Petri dishes lined with a cushion of 0.2%
agarose to prevent adhesion of the eggs (Klein and Jaffe, 1984) and aga-
rose plus 0.5% bovine serum albumin in FSW (Crawford, 2002).
However, agarose and BSA were not successful in obtaining chorion ex-
pansion for the oceanic squid Dosidicus gigas (Staaf et al., 2011).
2.3.4. Anaesthetic
Ethics and welfare laboratory protocols should be followed during
experimentation with live cephalopod embryos and paralarvae
resulting from fertilizing experiments (Mather and Anderson, 2007;
Moltschaniwskyj et al., 2007). Anaesthetic should be used to reduce
pain when necessary, particularly before sacrificing individuals. Mag-
nesium chloride (MgCl
2
) is an effective anaesthetic and narcotizing
agent for cephalopods, and over-anaesthesia can be used to euthanize
individuals (Messenger et al., 1985; Mooney et al., 2010). Alternatives
to MgCl
2
include chilling and increasing concentrations of ethanol
from 1 to 5%.
2.4. Step-by-step recipe for in vitro fertilization and egg incubation
2.4.1. Activation of spermatozoa
As noted before, spermatophores or spermatangia can be used as
sperm sources. Spermatophores can be collected by dissecting the
spermatophore sac of mature males. Spermatangia are located on ex-
ternal surfaces of the female body (see above) and sometimes may
have mud or organic debris. In this case, place whole spermatangia
on a 1-mm mesh and vigorously flush with seawater to remove or-
ganic debris from their surface before collecting the sperm. Cut
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sperm sources in small portions (in ex., ≤2 mm) on a Petri dish using
scissors or razor blades until a homogenized mass is obtained. For
spermatozoa activation, sperm should be in contact with FSW. Trans-
fer the chopped spermatophores or spermatangia to a glass container
and add FSW (optional step: FSW with ampicillin and streptomycin,
25 mg l
−1
) to obtain a milky sperm suspension that should be gently
agitated for 3–5 min. Filter this solution with 150 μm mesh to remove
debris of spermatophores or spermatangia. Add the sperm suspen-
sion to the oocytes within 25 min of spermatozoa activation
(Villanueva et al., 2011). Spermatozoa mobility can be checked
under a microscope at 200–400× magnification.
2.4.2. Collection of oocytes
For large oviducts, cut the membrane of the oviduct and extract
the oocytes from deep inside, using a sterile plastic spoon to avoid mi-
crobial infection (Sakurai et al., 1995). For small oviducts, cut the en-
tire oviduct, then pick up the pieces with tweezers and let the oocytes
drip out into sterile Petri dishes. In each dish, a small drop containing
about one to two hundred oocytes will be sufficient. Cover the dish
with a lid as soon as oocytes are deposited. Before the addition of
sperm, oocytes can remain some minutes virtually dry on the dish
(range 8 to 12 min), which does not affect their capacity to be fertil-
ized (Villanueva et al., 2011).
Optional step: Some species, such as T. pacificus, may need oocyte
hydration before fertilization. For hydration, oocytes need to be
placed in a container with seawater for a few minutes (Sakurai et
al., 1995).
2.4.3. Fertilization
Add drops of sperm suspension using a sterile pipette, enough to
cover the pile of oocytes. Carefully mix the oocytes and sperm sus-
pension with the tip of the pipette to homogenize the medium, avoid-
ing damage to the oocytes. A gamete contact time of 20–30 min
seems to be sufficient for fertilization. Temperature during the fertil-
ization process should be controlled. High room temperatures may
affect gamete quality and/or early embryo development, depending
on the species' natural temperature range.
Optional step: Oocytes collected from the oviduct, washed several
times in FSW and added directly to the sperm suspension resulted in
high fertilization rates in D. pealeii (Crawford, 2002).
Optional step: If the quantity of available sperm is limited, fertili-
zation success may be increased by using a longer contact time be-
tween gametes, as observed in fishes (Butts et al., 2009).
2.4.4. Egg incubation
After fertilization, add oviducal gland jelly to cover all the eggs,
filling approximately half of the Petri dish. Gently stir again. Transfer
dishes to incubators. Two to three hours after fertilization (depending
on temperature), successfully developing eggs should be identifiable
by one of several methods. Observation of cleavage is unambiguous.
In some cases, developing eggs can also be distinguished by chorion
expansion and a relatively spherical shape in comparison with the
ovoid shape of the unfertilized oocytes. At this point, successfully fer-
tilized eggs can be transferred with pipettes to new dishes at the de-
sired egg density. For incubations using FSW without antibiotics,
densities of 10–15 eggs per dish of 50 mm diameter can be incubated
at 22 °C (Sakurai et al., 1995). Sakai et al. (2004) reported egg incuba-
tions at temperatures ranging from 11.4 to 25.4 °C in polystyrene
multi-well plates with 6 wells of 35 mm diameter and 15 ml volume,
at a density of 20 eggs in 10 ml seawater (2 eggs ml
−1
). Seawater in
the wells was changed at least four times a day. When using antibi-
otics, a conservative egg density of ≤60 eggs per dish of 60 mm diam-
eter (≤5 eggs ml
−1
) and a daily change of seawater is recommended
at an incubation temperature of 17 °C (Villanueva et al., 2011).
Depending on the species, care should be taken when transferring
eggs. Mechanical stress imposed by aspiration of recently fertilized
eggs of Doryteuthis pealeii using microhematocrit pipettes does not
affect initial stages of development (Kao, 1985). However, Sakai et
al. (2011) showed that embryos of I. argentinus at developing stages
5 to 12 (from second cleavage until blastoderm stage; embryos aged
4.5 to 11.5 h after fertilization at 20 °C) were the most sensitive to
mechanical stress produced by aspiration, and caution should be
taken when handling eggs during this critical period. FSW (optional:
with antibiotics) can be replaced every day using sterile pipettes
until hatching. Dead embryos should be removed, and counted, if
necessary, to estimate mortality rates. During daily water changes,
take care to avoid temperature shocks, and never leave the eggs with-
out water. High room temperatures during water changes may affect
embryo development and survival. The experimental design should
include an adequate number of replicates for each treatment in
order to permit statistical analysis of the results.
Optional step: If necessary, after the addition of oviducal jelly, eggs
can remained more than 2–3 h in this medium until the first FSW
change, without apparent damage. For example, recently fertilized
eggs of I. coindetii maintained for 19 h in oviducal jelly +antibiotics
until the first FSW change and incubated at 17 °C resulted in a 44±
22 survival rate to hatchling (R. Villanueva, unpublished).
Optional step: Depending on the experimental objective, more ad-
ditions of oviducal jelly during embryonic development may be ap-
plied to increase chorion expansion. A second oviducal jelly addition
before organogenesis in I. coindetii embryos resulted in a larger egg
diameter, partially delayed hatching and heavier hatchling squids, in
comparison with treatments receiving only one jelly addition
(Villanueva et al., 2011).
3. Future research
The in vitro techniques described here are relatively simple and can
be used on oceanographic vessels and in land laboratories, but they
may require some improvement and adaptation to each different
squid species. In other groups of molluscs and fishes, experimental
techniques greatly influence fertilization success (Butts et al., 2009;
Song et al., 2009). Fertilization techniques in squids need to overcome
the previous lack of a standardized methodology and procedures will
benefit from further clarification of such important fertilization param-
eters as optimal oocyte concentration, seawater fertilization volume
and spermatozoa: oocyte ratio. Little progress has been made in eluci-
dating the motility of squid spermatozoa (Iwata et al., 2011;
Laptikhovsky, 1990; Laptikhovsky and Nigmatullin, 1996; Wang et al.,
2011) and the spermatozoa density necessary for suitable squid oocyte
fertilization has not been determined. Up to now an excess of sperm
has probably been used in order to guarantee fertilization success.
Methods to determine gamete quality and density, critical gamete
contact time and spermatozoa motility are well established in marine
fishes (see among others: Chereguini et al., 1999; Cosson et al., 2008;
Rurangwa et al., 2004; Suquet et al., 1995; Tvedt et al., 2001) and we
expect that many of these techniques can be adapted to squids. How-
ever, certain reproductive characteristics of cephalopods may make in
vitro fertilization easier than in fish. For example, the existence of a
spermatozoa attractant as observed in the cuttlefish Sepia officinalis
may facilitate fertilization by increasing chances of gamete contact,
and opens new experimental approaches in this field (Zatylny et al.,
2002). In addition, the biflagellate spermatozoa of Illex squid enable
them to swim at high velocity of 130–140 μms
−1
(Laptikhovsky
and Nigmatullin, 1996), which probably enhances gamete contact
and increases fertilization rates. This spermatozoa velocity is close
to the 167 μms
−1
recorded for Heterololigo bleekeri (Iwata et al.,
2011) and higher than that of other cephalopod species, most of
which fall between 25 and 65 μms
−1
(Laptikhovsky, 1990).
Fertilization in rare oceanic squid species may be facilitated in the
future through the conservation and storage of sperm by refrigeration
and cryopreservation. Naud and Havenhand (2006) showed that
130 R. Villanueva et al. / Aquaculture 342–343 (2012) 125–133

Author's personal copy
cuttlefish spermatophores refrigerated for two months at 4 °C still
contain mobile spermatozoa. Similar capabilities may be suspected
of squid sperm. Techniques for the cryopreservation of squid sperm
are available (Robles et al., submitted for publication) and promise
to be a useful tool for in vitro fertilization programs as in other aquatic
invertebrates and marine fishes (see among others: Bart et al., 2006;
Cabrita et al., 2010; Gwo, 2000; Vuthiphandchai et al., 2007), allowing
squid sperm availability throughout the year. Techniques for cephalo-
pod gamete preservation, particularly female gametes, should be ex-
plored. For example, chilled storage (2 °C) of unfertilized fish
oocytes packed into sealed polyethylene bags without a gas-filled
space resulted in 50% of fertilization rate after 20 days (Komrakova
and Holtz, 2011), and squid oocytes may have similar potential.
Premature hatching seems to be a common event in oceanic squid
fertilized in vitro (Sakai et al., 1998; Villanueva et al., 2011; Watanabe
et al., 1996; Yatsu et al., 1999). Watanabe et al. (1996) noted that eggs
from wild egg masses of T. pacificus hatched two stages later than ar-
tificially fertilized eggs and suggested that wild eggs were enwrapped
by nidamental jelly in addition to oviducal jelly, and this natural gelat-
inous matrix may delay hatching. Research on the function(s) of the
oviducal and nidamental glands in the formation of natural egg
masses and natural egg development will probably suggest improve-
ments to in vitro fertilization techniques in the near future. It is as-
sumed that the oviducal mucosubstance, which is essential for
chorion expansion in fertilized eggs, surrounds each egg when
spawned (Ikeda et al., 1993). Kimura et al. (2004) showed that the
water-soluble fraction of the mucosubstance of the nidamental gland
forms the egg mass surface layer and the insoluble fraction forms the
fibrils within the egg mass of T. pacificus egg masses. In the same spe-
cies, Ikeda and Shimazaki (1995) showed that nidamental gland jelly
does not induce the formation of perivitelline space at fertilization.
However, chorionic expansion was obtained in Abralia,Abraliopsis,
Sthenoteuthis and Thysanoteuthis by using freeze-dried nidamental
gland of Loligo sp. (Arnold and O' Dor, 1990) and in Doryteuthis pealeii
by adding a cushion of 0.2% agarose (Klein and Jaffe, 1984) or agarose
plus 0.5% bovine serum albumin (Crawford, 2002). These different re-
sults suggest that probably more than one factor contribute to chori-
onic expansion, and further research is needed.
The use of antibiotics (ampicillin and streptomycin) to avoid mi-
crobial infections is recommended because their experimental use
seems to produce higher embryo survival rates. However, the exis-
tence of secondary effects of these antibiotics on the embryo is un-
known and different antibiotics and concentrations should be
explored. Antimicrobial activity of the accessory nidamental gland
and egg cases has been detected in loliginid squid and may inhibit mi-
crobial growth on the egg capsule through several mechanisms, such
as reducing ciliary activity (Atkinson, 1973; Barbieri et al., 1997; Biggs
and Epel, 1991). This antimicrobial activity has been attributed to the
higher levels of unsaturated fatty acids present on accessory nida-
mental glands of ripe individuals (Gomathi et al., 2010), suggesting
that the accessory nidamental gland may help loliginid embryos to
resist infection. Although oegopsid squid lack these accessory glands
(Young et al., 1998) the oegopsids nidamental glands should be stud-
ied for antimicrobial properties.
To date, all incubations of embryonic squid have been done at a
small scale, using Petri dishes. Investigations into larger egg incuba-
tion systems will help to reduce working time and facilitate the col-
lection of large numbers of hatchlings. Furthermore, larger scale
systems may permit the rearing of oceanic squid paralarvae, which
has not yet been successful for any species.
4. Conclusions
Egg masses of oegopsid squid are spawned in the open ocean,
making them difficult to detect. In view of the challenges involved
in acquiring naturally spawned eggs, in vitro fertilization techniques
have provided an alternative method for obtaining valuable informa-
tion on the eggs and paralarvae of oceanic squids. A laboratory guide
for the in vitro fertilization of oceanic squid is provided here with the
aim of recommending laboratory materials, sources and preservation
of gametes, and improving existing methods for fertilization and egg
incubation. Adequate egg chorion expansion and premature hatching
are identified as major challenges for in vitro fertilization. Future re-
search on the functions and properties of the oviducal and nidamen-
tal glands of the mature females will probably suggest improvements
to obtain suitable egg chorion expansion, likely reducing premature
hatchling. Methods to determine gamete quality should be estab-
lished as well as procedures to determine important fertilization pa-
rameters such as optimal gamete density, critical gamete contact
time and spermatozoa: oocyte ratio. Development of new tools, in-
cluding the preservation of gametes by refrigeration and cryopreser-
vation, will enhance future studies. In vitro fertilization techniques
will continue to be a necessary step to develop the larval culture of
this group of cephalopods and essential in obtaining comparative ma-
terial for paralarval taxonomy, as well as opening possibilities for the
culture of oceanic squid —so ecologically critical, and yet still so poor-
ly understood.
Acknowledgments
We would like to acknowledge CIBNOR and the organizers of the
5th International Symposium on Pacific Squid for the stimulating
days spent in La Paz, Mexico, which resulted in the preparation of
this manuscript. RV was funded by the research project CALOCEAN
(AGL2009-11546) from the Ministry of Science and Innovation of
Spain and RdeS by COFAA and EDI grants.
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