Batchwise assessment of porcine embryos for cryotolerance

Fujino, Yukihiro; Kikuchi, Kazuhiro; Nakamura, Yoshiyuki; Kobayashi, Hiroshi; Yonemura, Isao; Suzuki, Misae; Misumi, Koji; Nagai, Takashi

doi:10.1016/j.theriogenology.2006.08.008

Cited by 9 publications

(12 citation statements)

References 28 publications

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“…Traditional slow freezing has a very low effectiveness in pig embryos [1,2], although some success had been reported since the late 1980s [3][4][5]. A remarkable breakthrough to increase the efficacy of cryopreservation of porcine embryos was reported by Nagashima et al [6,7], who found that mechanical removal of intracellular lipid (a method referred to as ''delipation'') in early-stage embryos significantly increased their tolerance to chilling and cryopreservation.…”

Section: Introductionmentioning

confidence: 99%

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Somfai

Ozawa

Noguchi

et al. 2009

Biology of Reproduction

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We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.

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Section: Introductionmentioning

confidence: 99%

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Somfai

Ozawa

Noguchi

et al. 2009

Biology of Reproduction

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“…Nevertheless, since survival and development of vitrified porcine zygotes (having the same size and lipid content as oocytes) is significantly higher than those of vitrified mature or immature oocytes [11,12], ultra-structural characteristics specific to those unfertilized oocytes should also be considered as potential reasons for their low cryotolerance. In pigs, traditional freezing could be successfully applied for semen [5] and even for in vivo produced embryos [13][14][15]; however it does not seem to work well for oocytes [16]. The first success in cryopreservation of porcine oocytes (i.e.…”

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Factors Affecting Cryopreservation of Porcine Oocytes

2012

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“…The rates of transferred embryos that survived to the piglet stage were calculated as survival rates [11][12][13][14][15].…”

Section: Pregnancy Diagnosis and Farrowingmentioning

confidence: 99%

“…In addition to farrowing rate and litter size, we also evaluated survival rate [11][12][13][14][15], as this seems be a useful indicator of transfer efficiency.…”

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confidence: 99%

Effect of Volume of Non-Surgical Embryo Transfer Medium on Ability of Porcine Embryos to Survive to Term

Nakazawa¹,

Misawa²,

Fujino

et al. 2008

Journal of Reproduction and Development

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Abstract. Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 µm in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 ± 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium. Key words: Embryo survival, Medium volume, Non-surgical transfer, Pig (J. Reprod. Dev. 54: [30][31][32][33][34] 2008) pioneering study demonstrated that it is possible to transfer porcine embryos non-surgically using a double-walled polythene catheter and obtain successful pregnancy [1]. Thereafter, many researchers succeeded in producing viable piglets after transfer of in vivo-derived embryos using both a commercially available artificial insemination (AI) catheter [2-4] and a specially designed flexible catheter [5][6][7]. This technique offers considerable promise for improving the efficiency of embryo transfer in the pork industry and also for biotechnology applications. However, the technique still has drawbacks because of the low survival rate of transferred embryos and the variable degree of successful transfer.It has been suggested that there are two important parameters related to the success of non-surgical embryo transfer: (1) the embryo deposition site [8] and (2) the volume of transfer medium associated with the embryos [9]. AI catheters require much more medium-10 to 50 ml-when used as a transcervical catheter [3,4]. To allow the transfer of embryos with small amounts of fluid, several types of flexible catheter have been developed. In an experiment by Hazeleger and Kemp [5] and a subsequent study by the same research group [10], embryos were...

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Batchwise assessment of porcine embryos for cryotolerance

Cited by 9 publications

References 28 publications

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Factors Affecting Cryopreservation of Porcine Oocytes

Effect of Volume of Non-Surgical Embryo Transfer Medium on Ability of Porcine Embryos to Survive to Term

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