In-vitro fertilization of pig oocytes by frozen boar spermatozoa

Nagai, Takashi; Takahashi, T.; Masuda, Hiroshi; Shioya, Yasuo; Kuwayama, M.; Fukushima, Mai; Iwasaki, Setsuo; Hanada, Akira

doi:10.1530/jrf.0.0840585

Cited by 156 publications

(102 citation statements)

References 7 publications

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“…Causes for this problem likely include the quality of semen at fertilization (Kim et al, 1996a;Pavlok, 2000;Sirard et al, 1993). Large variations among individual males have been noted in fertilization rates with fresh (Sirard et al, 1993) and frozen sperm (Nagai et al, 1988). Further, because the difficulty of cryopreservation of boar semen, little information is available on the relative penetration rates of fresh and frozen boar sperm from a single ejaculate.…”

Section: Introductionmentioning

confidence: 99%

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

Suzuki

Saito

Kagawa

et al. 2003

Microscopy Res & Technique

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Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a total of 15 boars of three different breeds, revealed that the inter-breed variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also shown that the incidence of polyspermy as well as penetration rate was greatly decreased by freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment, and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule domains in association with individual male pronuclei (PN), but in a high degree of polyspermy (more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of paternal and maternal origin on the cytoskeletal reorganization is also discussed.

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Section: Introductionmentioning

confidence: 99%

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

Suzuki

Saito

Kagawa

et al. 2003

Microscopy Res & Technique

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“…66(1): [37][38][39][40][41] 2004 Sperm reach maturation during migration from the caput to the cauda of the epididymis and are retained in the cauda epididymis until ejaculation. It has been demonstrated in many species that sperm in the cauda epididymis exhibited fertility in in vitro fertilization and artificial insemination [1,4,[7][8][9]14]. For animals that die unexpectedly, transmigration of epididymal sperm, and its freeze-storage are important techniques for gamete preservation.…”

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confidence: 99%

Artificial Insemination of Frozen Epididymal Sperm in Beagle Dogs

Hori

Ichikawa

Kawakami

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 ± 1.6 (SE) %; sperm viability, 89.1 ± 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 × 10 7 , mean 61.5 ± 10.0 × 10 7 . Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 ± 2.5% and 53.1 ± 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 × 10 8 , 3 × 10 8 , or 4 × 10 8 sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 × 10 8 sperm was fertilized (1/16, 6.3%). When 1 × 10 8 sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate. KEY WORDS: canine, epididymal sperm, frozen, intratubal insemination, intrauterine insemination.J. Vet. Med. Sci. 66(1): [37][38][39][40][41] 2004 Sperm reach maturation during migration from the caput to the cauda of the epididymis and are retained in the cauda epididymis until ejaculation. It has been demonstrated in many species that sperm in the cauda epididymis exhibited fertility in in vitro fertilization and artificial insemination [1,4,[7][8][9]14]. For animals that die unexpectedly, transmigration of epididymal sperm, and its freeze-storage are important techniques for gamete preservation. These techniques are considered important for preventing species from becoming extinct. Conception obtained by artificial insemination of frozen epididymal semen has been reported in one dog [7], pigs [8], goats [1], and cats [14]. Marks et al. [7] successfully obtained delivery of one pup after intrauterine insemination despite poor semen quality after thawing. There is only one study about the quality and resistance to freezing of canine epididymal sperm, reported by Hewitt et al. [5]. Their study showed that the number of sperm that reached maturation was lower in the epididymis than in frozen ejaculated sperm, but there was no difference in oocytepenetrating ability [5]. In the method of epididymal sperm recovery, Marks et al. [7] reported that the epididymis was perfused from the seminal duct to the epididymis in boxer dogs and Hewitt et al. [5] used the mincing method. Sirivaidyapong [11] com...

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“…It is suggested that spermatozoa in epididymides during refrigeration retain minimum ability for fertilization, which differs depending on the boar as with ejaculated spermatozoa. Boar epididymal spermatozoa can be frozen and used for IVF [2]. Normally, before freezing, they are diluted with extenders at a temperature of between 15 C and 4 C, then gradually cooled to 4 C and cryopreserved, using the same procedure as for ejaculated boar spermatozoa (reviewed by Bwanga [14]).…”

Section: Discussionmentioning

confidence: 99%

“…After thawing at 37 C, the spermatozoa were preincubated for 1 h in Medium 199 which had been adjusted to pH 7.8 [2]. A portion (10 µl) of preincubated sperm was introduced into 90 µl fertilization medium, Bracket and Oliphant solution [13] supplementing with bovine serum albumin (BSA; Fraction V, Sigma) (10 mg/ml), caffeine (Sigma) (5 mM) and casein phospho peptide (Meiji Seika Kaisha, Ltd., Tokyo, Japan) (1 mg/ml) containing 10 IVM oocytes surrounded by expanded cumulus cells.…”

Section: Ivm Ivf and Etmentioning

confidence: 99%

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Reproduction in Pigs Using Frozen-Thawed Spermatozoa from Epididymis Stored at 4C.

Kikuchi

Kashiwazaki

Nagai

et al. 1999

Journal of Reproduction and Development

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Abstract. The ability of frozen-thawed boar spermatozoa obtained from epididymides stored at 4 C for 1 day to produce piglets after Fallopian insemination or in vitro fertilization (IVF) of in vitro matured (IVM) oocytes was examined. To test their in vivo fertilization ability, frozen-thawed spermatozoa from 2 boars, which had already shown IVF ability, were introduced into the Fallopian tubes of estrus-synchronized gilts. Embryo collection on the 7th day post-insemination revealed that one of two batches of spermatozoa had an ability of fertilization in vivo and that fertilized oocytes could develop to blastocysts. Three of 6 gilts inseminated with one batch of spermatozoa became pregnant and one of them farrowed 2 normal piglets (1 male and 1 female). IVM oocytes fertilized in vitro by the same spermatozoa were transferred to 3 recipients (200 oocytes per recipient). One of them became pregnant and farrowed 5 normal piglets (3 males and 2 females). The results indicate that boar spermatozoa collected from epididymides stored for 1 day at 4 C and then frozen have the ability to produce piglets. The new cryopreservation protocol and reproductive technology described here can enhance conservation of boar genetic resources when the collection site of the epididymides is far from a laboratory.

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In-vitro fertilization of pig oocytes by frozen boar spermatozoa

Cited by 156 publications

References 7 publications

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

Artificial Insemination of Frozen Epididymal Sperm in Beagle Dogs

Reproduction in Pigs Using Frozen-Thawed Spermatozoa from Epididymis Stored at 4C.

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