Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts

doi:10.1016/S0093-691X(02)01163-9

Theriogenology

Volume 59, Issues 5–6, March 2003, Pages 1393-1402

https://doi.org/10.1016/S0093-691X(02)01163-9 Get rights and content

Abstract

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1) medium only control; (2) activin (10 ng/ml); (3) epidermal growth factor (EGF) (10 ng/ml); (4) insulin 5 μg/ml and (5) GH (100 ng/ml). There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0±1.5 and 93.1±3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3±3.4 and 62.0±3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7±3.3%. With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.

Introduction

Numerous agricultural and biomedical applications are emerging from our steadily advancing ability to preserve and manipulate gametes and embryos of domestic animals. Cryopreservation of embryos produced in vivo or in vitro is a key factor in bovine embryo transfer technology and other emerging embryo technologies. However, survival rates of cryopreserved in vitro produced embryos, as measured either by post-thaw survival in culture or by pregnancies after transfer, has been lower than those reported for in vivo derived embryos [1]. Also, embryos produced in vitro or pre-exposed to in vitro culture conditions have been shown to be more sensitive to cryoinjury than those produced in vivo [1], [2]. Recently, the efficacy of cryopreservation for in vitro produced (IVP) embryos appears to have been improved by changes in the culture systems and the method of cryopreservation. Removal of serum from the medium for culturing presumptive bovine zygotes has been shown to improve the resistance of blastocysts to deleterious effects of cryopreservation [3], [4]. In addition, the negative effects of serum culture on subsequent development have also been reported [5], [6]. Hochi et al. [7] demonstrated that linoleic acid-albumin added to the culture media increased the survival of bovine embryos in vitro after cryopreservation. An accelerated rate of cooling has been demonstrated to overcome chilling sensitivity of bovine oocytes and embryos in vitro [8], [9].

Growth factors and cytokines may be viewed as local regulators involved in the subtle coordination of cellular proliferation and differentiation. Insulin has been shown to increase the rate of glucose transport in blastocysts [10] and to decrease apoptosis and increase cell proliferation [11]. Culture of bovine cumulus oocyte complexes (COCs) in the presence of growth hormone (GH) accelerates the process of nuclear maturation and increases cumulus expansion, rate of cleaved embryos and of blastocysts due to the improved distribution of cortical granules [12], [13]. Epidermal growth factor (EGF) stimulates distinct cellular functions, which suggests a possible effect on early development of mammalian embryos [14]. In early preimplantation embryos, it has been noted that activin (recombinant human activin-A) stimulates the development and early preimplantation of embryos to the blastocyst stage when they are cultured in a chemically defined medium [15], [16].

In our previous report [17], we successfully employed a pre-frozen straw vitrification procedure to enhance survival and development of blastocysts. The advantage of this method over the open pull method [9] is that it makes possible direct transfer of the embryos to the recipient. Therefore, the purpose of the present study was to investigate the influence of specific growth factors and GH in the culture medium on in vitro maturation (IVM), in vitro fertilization (IVF), IVP and post-thaw survival of vitrified blastocysts.

Section snippets

Ovary collection, oocyte selection, and IVM

Bovine ovaries were collected from Japanese Black cows at a local abattoir and were transported within 3 h to the laboratory in Ringer’s solution supplemented with penicillin-G (100 IU/ml) and streptomycin sulphate (0.2 μg/ml) at 30–32 °C in a thermos flask. The COCs were recovered by aspiration of 2–8 mm follicles and selected based on the presence of a multi-layered compact cumulus investment. Selected COCs were rinsed in the basic maturation medium which consisted of HEPES buffered TCM199

Effect of growth factors and GH on nuclear maturation

A total of 1673 oocytes were used for checking the nuclear status of oocytes after in vitro culture in MM (n=560) or for IVF (n=1113). As shown in Table 1, there was an increase (P<0.05) compared to the controls in the percentage of oocytes that reached meta phase II when activin, EGF and GH were added to the maturation media. The addition of insulin did not result in any significant difference over controls. There was no significant difference between growth factor supplemented groups and GH

Discussion

The results of the present study clearly demonstrate that the post thaw development of in vitro produced vitrified embryos can be greatly improved by the addition of growth factors and GH. We were able to demonstrate that culture factors can accelerate and favor the development of thawed blastocysts to the hatched blastocyst stage as well as increase the total cell number per embryo.

Autocrine secretion of growth factors and GH by embryos and expression of specific receptors at particular stages

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¹: Permanent address: Faculty of Agricultural Sciences, University of British Columbia, Vancouver, Canada.

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Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts

Abstract

Introduction

Section snippets

Ovary collection, oocyte selection, and IVM

Effect of growth factors and GH on nuclear maturation

Discussion

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Cryobiology

Prog. Growth Fact. Res.

Theriogenology

Theriogenology

Cryobiology of in vitro-derived bovine embryos

Theriogenology

Cryopreservation of in vitro-derived bovine embryos produced in a serum-free culture system

Theriogenology

Container-less vitrification of mammalian oocytes and embryos

Nat. Biotech.

Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos

Mol. Reprod. Dev.

Insulin increases cell numbers and morphological development in mouse preimplantation in vitro

Reprod. Fertil. Dev.

Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis

Biol. Reprod.