20

Journal of Applied Animal Science Vol.11 (Supplement) 2018

Cryopreservation of gametes and gonadal tissues for
porcine genetic resources
Kazuhiro Kikuchi1,2,*, Hiroyuki Kaneko1, Michiko Nakai1, Tamas Somfai3,
Naomi Kashiwazaki4, Takashi Nagai5
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba 305-8602, Japan,
2
The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, 753-8511, Japan,
3
Institute of Livestock and Grassland Science, NARO, Tsukuba 305-0901, Japan,
4
Graduate School of Veterinary Science, Azabu University, Sagamihara, 252-5201, Japan,
5
Past address: Headquarter, NARO, Tsukuba, 305-8517, Japan

1*

Corresponding author: kiku@affrc.go.jp

Keywords: pig, cryopreservation, vitrification, oocyte, gonadal tissue, gene bank

Abstract

oocytes or early embryos has been considered

The conservation or preservation of mammalianchallenging. However, the utilization of female gametes

genetic resources, especially farm animals, has been

and gonadal tissues has been considered very important.

conducted under in situ conditions by maintaining

Some attempts at vitrification of tissues have been

living individuals as "livestock". However, systems for

made, and for this purpose, in vitro embryo production

laboratory in vitro embryo production using gametes(IVP) and its related technologies are essential.

such as spermatozoa and oocytes are now available and

The basic IVP procedure for piglet production

in vitro culture for embryos viable to offspring after

has been of fundamental importance in studies of embryotransfer to recipients, as an ex situ preservation method

freezing/vitrification at the pronucleus (Somfai et al.

for mammalian genetic resources. In pigs, freezing of sperm

2009), the 4- to 8-cell (Nagashima et al., 2007), the morulais the most reliable and well-established method for this(Maehara et al., 2012), and the blastocyst (Mito et al.,purpose. On the other hand, cryopreservation of female2015) stages. Intracytoplasmic sperm injection (ICSI)-

gametes (oocytes) and gonadal (testicular and ovarian)

related technologies (Nakai et al., 2003, 2009, 2010,

tissues usually by vitrification has been conducted;

Kaneko et al., 2013) are also fundamental for the

however, resulted in very low efficacies. Recently, ourutilization sperm without self-penetration ability into

laboratory conducted some research themes related tooocytes.

these issues. We have been focusing on advances using
porcine in vitro embryo production (IVP) systems andCryopreservation of unfertilized oocytes

xenografting into host immunodeficient mice for piglet

Freezing of spermatozoa is one of the basic

generation. In this symposium, we introduce recent

approaches for preservation of genetic resources. Oocyte

progress on the vitrification of porcine immature

cryopreservation is a basic strategy for gene banking ofoocytes and gonadal tissues followed by their IVP and

female germplasm; for pigs. However, this technology

xenografting to produce gametes.

is very difficult and still considered a challenge.
We have tested the possibility of vitrification for

Introduction

immature (at the germinal vesicle stage; cumulus oocyte

The main type of genetic material for routine

complexes) and mature (at the metaphase-II stage) oocytes

cryopreservation by gene banking is spermatozoa, so called

before fertilization (Somfai et al., 2012). The survival ratesemen cryobanking. Banking of other materials such as

for mature oocytes was relatively high; however, a high

Journal of Applied Animal Science Vol.11 (Supplement) 201821

proportion (about 70%) of vitrified immature oocytes waswere obtained after 4 months. Just after collection, someable to complete meiotic maturation and undergo normal

spermatozoa showed only faint motility for a short period;

fertilization and embryo development. Using cryopreserved

however, they could not penetrate into IVM oocytes,

immature oocytes, we had focused on piglet production

meaning that we unfortunately could not apply theseafter in vitro maturation/fertilization (IVM/IVF) andsperm for IVP and have to select ICSI method to obtainembryo transfer, and finally obtained piglets (Somfai

fertilized oocytes. After culture, some of them developed

et al., 2014).

into blastocysts; which were similar to the oocytes from

This application has been confirmed using

prepubertal gilts after IVM/IVF (Kikuchi et al., 2002,

immature oocytes from commercial Western pigs, which

Nakai et al., 2009]. When oocytes at the pronuclear

seems to be also efficient reproduction system in native

stage after ICSI were transferred to oviducts ofspecies especially, in Asian countries. We have conductedestrous-synchronized recipients, we were able to obtainunder the project by Science and Technology Researchboth male and female piglets (Nakai et al., 2010), which

Partnership for Sustainable Development (SATREPS) for

showed normal reproductive abilities when developed

Vietnamese native pigs (https://www.jst. go.jp/global/

to the adulthood (Kaneko et al., 2012).

english/kadai/h2604_vietnam.html) and found equal

For more advanced utilization of this technique,efficacy in cryopreservation efficacies (Somfai et al.,we have investigated the possibility of vitrification of

unpublished data).

testicular tissue fragments before xenografting. Thismethod enables long term storage in liquid nitrogen of the

Cryopreservation of gonadal tissues
1) Testicular tissue

tissue and the production of sperm whenever the needarises. After a certain period (120 days), sperm were first

One approach for inducing spermato-genesis

recovered from the grafts. The sperm recovery rate

in isolated testicular tissues is ectopic (into another site inincreased with time after grafting from 180 to 350 days.

the body) grafting into immunodeficient host animals

Sperm after the recovery from the tissues were applied(xenografting). Ectopic xenografting has been reported

for ICSI, and the oocytes were transferred to recipients.

for hamsters (Schlatt et al., 2002), goats (Honaramooz

We could obtain live male and female piglets (Kaneko

et al., 2002), rhesus macaques (Honaramooz et al., 2004),

et al., 2013). Both the male and female pigs showed

sheep (Zeng et al., 2006), cats (Snedaker et al., 2004), andnormal reproductive abilities (Kaneko et al., 2014).

pigs (Nakai et al., 2009, Nakai et al., 2010, Honaramooz

2) Ovarian tissues

et al., 2002, Zeng et al., 2006, 2007, Honaramooz et al.,Primordial follicles act as stores for ovarian

2008, Kaneko et al., 2008). It is preferable to graft

follicles and can be potential sources of oocytes. To utilize

testicular tissue under the skin of the back of commerciallythese resources, ovarian tissue grafting is a possible method

available severe combined immunodeficiency mice orfor growing or maturing such primordial oocytes in thenude mice.

primordial follicles of large mammals. In humans andA series of studies has been conducted in our

primates, grafting of ovarian tissues to another site in thelaboratory (Nakai et al., 2009, Nakai et al., 2010, Kaneko

body (autografting) has been used successfully to produce

et al., 2008, Kikuchi et al., 2011) to evaluate whether

viable offspring (Lee et al., 2004, Donnez et al., 2004).

boar spermatogonia can develop into sperm in testicular

Xenografting is more advantageous for the conservation

tissues grafted into immunodeficient nude mice, andand multiplication of domestic or endangered animals.

whether live piglets can be produced from this sperm bySome studies have been conducted in cat (Gosden et al.,ICSI into oocytes. Elongated spermatids and spermatozoa

1994), sheep (Gosden et al., 1994), mice (Snow et al.,

22

Journal of Applied Animal Science Vol.11 (Supplement) 20182002), humans, (Oktay et al., 1998, Weissman et al., 1999,

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