Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in Bombyx mori.

Abstract

Female moths of Bombyx mori were artificially inseminated with cryopreserved semen. The fertility of inseminated females varied from 0% to 76.9% depending on the strain. Addition of fresh semen from triploid males, which are infertile but whose semen includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from normal males. Frozen apyrene sperm from the triploid donors also improved the fecundity of females, inseminated with cryopreserved normal semen, but less than fresh semen from triploid males. Fertilization success in B. mori requires the presence of both, intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the cryopreservation process better than apyrene sperm. Hence, we recommend to add apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved semen in artificial insemination. This may advance the application of cryopreservation as a routine technique to maintain silkworm resources. The technique may also be applicable to other moth and butterfly species which, like B. mori, possess eupyrene and apyrene sperm.

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Title
Apyrene sperm from the triploid donors restore fecundity of
cryopreserved semen in Bombyx mori
Author(s)Takemura, Yoko; Sahara, Ken; Mochida, Yuji; Ohnuma, Akio
CitationJournal of Insect Physiology, 52(10): 1021-1026
Issue Date2006-10
Doc URLhttp://hdl.handle.net/2115/16978
Right
Typearticle (author version)
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Information
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Apyrene sperm from the triploid donors restore fecundity of cryopreserved semen in
Bombyx mori
Yoko Takemuraa, Ken Saharab*, Yuji Mochidaa and Akio Ohnumaa

a Institute of Sericulture, 1053 Iikura, Ami-machi, Ibaraki 300-0324, Japan
b Laboratory of Applied Molecular Entomology, Graduate School of Agriculture,
Hokkaido University, Sapporo 060-8589, Japan

*To whom Correspondence should be addressed,
Dr. Ken Sahara

Fax: +81-11-706-2402. Tel: +81-11-706-2402
E-mail: sahara@abs.agr.hokudai.ac.jp
Phone & Fax: 011-706-2402

Running title: Cryopreserved sperm in Bombyx mori

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Abstract
Female moths of Bombyx mori were artificially inseminated with cryopreserved semen.
The fertility of inseminated females varied from 0% to 76.9% depending on the strain.
Addition of fresh semen from triploid males, which are infertile but whose semen
includes intact apyrene sperm, greatly improved fecundity of cryopreserved semen from
normal males. Frozen apyrene sperm from the triploid donors also improved the
fecundity of females, inseminated with cryopreserved normal semen, but less than fresh
semen from triploid males. Fertilization success in B. mori requires the presence of both,
intact eupyrene and apyrene sperm. Our results show that eupyrene sperm tolerate the
cryopreservation process better than apyrene sperm. Hence, we recommend to add
apyrene sperm from the triploid donors as helper sperm routinely to cryopreserved
semen in artificial insemination. This may advance the application of cryopreservation
as a routine technique to maintain silkworm resources. The technique may also be
applicable to other moth and butterfly species which, like Bombyx mori, possess
eupyrene and apyrene sperm.

Key words: apyrene sperm; artificial insemination; cryopreservation; eupyrene sperm;
silkworm

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1. Introduction

Cryopreservation of sperm and artificial fertilization and/or artificial insemination serve
practical and research purposes in animals (Polge and Rowson, 1952; Graham et al.,
1978; Polge, 1978). In insects, methodological progress in this field may reduce work
cost and laboratory space in genetically important species such as Drosophila
melanogaster, Apis mellifera and Bombyx mori.
B. mori is not only genetically but also economically important. More than 3,000
strains (Yamamoto, 2000) and 400 mutations are known which have to be maintained
by rearing year after year. Recently, a feasible artificial insemination technique has been
developed in this species (Takemura et al., 1996; 1999), and successfully applied to
some races and hybrids using cryopreserved semen (Takemura and Kanda, 1999;
Takemura et al., 2000). This would open the gate to the frozen storage of silkworm
genetic resources without rearing the strains every year. The fertility of cryopreserved
semen and the number of fertilized eggs, however, varies depending on the race used
(Takemura and Kanda, 1999). Unfortunately, there are even races that do not tolerate
routine cryopreservation at all (this study).
Semen dissected from the vesicula seminalis contains eupyrene sperm bundles

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(fertilizing) and apyrene spermatozoa (non-fertilizing but indispensable for fertilization;
Osanai et al., 1988; Sahara and Kawamura, 2002, 2004; Sahara and Takemura, 2003;
also see review: Friedländer et al., 2005). Here we investigate the question of which
type of sperm is affected by the freezing and thawing process. We set out to develop a
more reliable method of artificial insemination using cryopreserved semen. This study,
therefore, may contribute to the establishment of a widely applicable, long-term,
silkworm semen cryopreservation and artificial insemination protocol.

2. Materials and methods

2.1. Bombyx mori strains
The B. mori L. strains used were Pure Mysole, Uryu, re9, Tw1, p50 and hybrids of
Hitachi × Nishiki and W2. re9 is a red egg (re/re) and stripe larval skin (pS/pS) mutant.
Tw1 and W2 are white egg (w-2/w-2) and plane (p/p) larval skin mutants. The other
strains all yield fertile eggs with a wild-type (black) color (+w-2/+w-2, +re/+re). p50 is a
regional Chinese strain, which was used in whole genome shotgun sequencing (Mita et
al 2004). Pure Mysole and Uryu are regional Indian and Chinese strains respectively,
and Hitachi × Nishiki is a practical hybrid for silk production. Since wild-type (black)

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