Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus)

Abstract

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.

Introduction

The common marmoset (Callithrix jacchus), a small New World primate originating from northeastern Brazil, is being increasingly used as a nonhuman primate (NHP) model in the field of biomedical sciences, including neuroscience, stem cell research, drug toxicology, immunology, and reproductive biology. Use of the marmoset has several advantages, including its small body size, prolificacy, and strong genetic similarity to humans [1].

Adult marmosets range in height from 25 to 35 cm and weigh approximately 350 to 500 g. The animal's small size offers advantages, such as low feeding costs, easy handling and retention, and relatively small breeding space requirements. Importantly, marmosets have several features facilitating the study of reproductive and developmental biology, such as early sexual maturation (12 to 18 mo), a relatively short gestation period (approximately 144 days), and postpartum ovulation and conception. Furthermore, the female marmoset has high fecundity; females usually give birth to twins and may produce 40 to 80 offspring during their lifetime. Additionally, the marmoset's ovarian cycle can be synchronized using prostaglandins, facilitating time- and cost-efficient embryo collection in a group of animals [2], [3]. Collectively, these advantages render the marmoset a highly suitable species for production of genetically modified nonhuman primates for human disease models. In that regard, transgenic marmosets carrying the green fluorescent protein gene with germ line transmission have been produced from naturally mated fertilized embryos and in vitro-fertilized embryos [4]. Numerous marmoset embryos are required to propagate this transgenic marmoset production technique and clarify marmoset embryonic development.

An efficient abdominal incision method for embryo collection in marmosets has been developed, but this method is invasive and animals required at least 2 mo for recovery [5]. A nonsurgical method has also has been described, but was difficult to reproduce [6]. Therefore, a new method for minimally invasive embryo collection is desirable. The overall objective of this study was to develop a new minimally invasive technique (transabdominal method) for collecting naturally conceived marmoset preimplantation embryos and to compare it with a more invasive technique (standard abdominal incision method) for collecting naturally conceived embryos from common marmosets. To assess the reproducibility of the newly established method, its applicability and success rate (i.e., embryo recovery) were tested at two research centers.