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. 2018 Aug 8:6:e5420.
doi: 10.7717/peerj.5420. eCollection 2018.

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring

Xiuzhi Tian #  1   2 , Dongying Lv #  1 , Teng Ma  1 , Shoulong Deng  1   3 , Minghui Yang  1 , Yukun Song  1 , Xiaosheng Zhang  4 , Jinglong Zhang  4 , Juncai Fu  1 , Zhengxing Lian  1 , Shien Zhu  1 , Yingjie Wu  1 , Yiming Xing  5 , Guoshi Liu  1
Affiliations

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring

Xiuzhi Tian et al. PeerJ. .

Abstract

Background: The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.

Methods: Ovine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.

Results: No significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.

Conclusions: The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

Keywords: AANAT; OPS vitrification; Pronuclear microinjection; Sheep.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. The structure of pBC1-AANAT plasmid.
Figure 2
Figure 2. Pictures of embryos before and after vitrification.
(A) Microinjected embryos before vitrification; (B) microinjected embryos after thawing.
Figure 3
Figure 3. The PCR detection of transgenic offspring.
M, marker, 1–13 represents transgenic offspring.
Figure 4
Figure 4. The sequencing detection of transgenic offspring.
Figure 5
Figure 5. Serum FSH, LH, E2, GnRH, and MT were measured in AANAT+ and AANAT− females.
AANAT+: AANAT transgenic-positive females; AANAT−: AANAT-negative females. (A–E) Serum production of FSH, LH, E2, GnRH, and MT collected at 0, 12, 24, 36, 48, and 60 h after vaginal CIDR removal from the control and transgenic-positive females. The times of 12, 36, and 60 h were at night. *Represents significant differences, P < 0.05.
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