Elsevier

Theriogenology

Volume 83, Issue 8, May 2015, Pages 1304-1309.e2
Theriogenology

Research article
Development of a modified artificial insemination technique combining penile vibration stimulation and the swim-up method in the common marmoset

https://doi.org/10.1016/j.theriogenology.2015.01.017Get rights and content

Abstract

The common marmoset, Callithrix jacchus, is used as a New World monkey species in biomedical studies because of its small body size and good reproduction in captivity. A modified artificial insemination technique was developed in this species to encourage breeding of lines carrying interesting genes and traits. Fresh semen was collected by penile vibratory stimulation. Medium containing highly motile sperm was inseminated into the uterus using a catheter. Seven females were inseminated using freshly prepared sperm from different males every day for 3 days including the expected ovulation day. As a result, four females conceived, and three females delivered six offspring in total (two singletons and one quadruplet). The paternity of the newborns was determined using microsatellite markers to accurately pinpoint the timing of insemination and ovulation. It is expected that our artificial insemination protocol can be effectively used to establish marmoset lines and genetically manage marmoset colonies.

Introduction

The common marmoset (hereafter marmoset) is a nonendangered New World monkey that has advantages as an experimental animal over other endangered primates, including small body size, early sexual maturity (approximately 1.5 years of age in both sexes), easy handling, and a nonseasonally breeding. These advantages make the marmoset a good model of nonhuman primates (NHPs) to investigate inheritable traits or phenotypes related to human disease [1].

However, there are two problems with marmoset reproduction. The first is that marmosets have definite mating preferences, as marmosets frequently display incompatible sexual behavior when paired for mating [2], [3]. Therefore, it is difficult to obtain offspring by crossing individuals with interesting traits or genes. Second, hand-reared males failed to copulate with other NHP including marmosets [4], [5], [6], [7]. We expect that these difficulties can be overcome using artificial insemination (AI) and assisted reproductive technology.

Marmosets are a good NHP model of reproductive biology to establish an AI technique because information on their reproductive endocrinology is available, ovulation can be confirmed retrospectively by measuring plasma progesterone (PRG) concentration [8], and ovulation can be controlled using the PGF2α analogue, cloprostenol [9]. In addition, four semen collection methods have been reported, including vaginal washing (VW) after copulation [3], electroejaculation (EE) [10], cauda epididymis (CE) [11], and penile vibratory stimulation (PVS) [12], [13]. Vaginal washing, EE, and CE have been used for AI [14], [15], [16]. Those procedures have advantages and disadvantages. Although VW is repeatable and has a high AI success rate (100%), it is an unreliable method when there is behavioral incompatibility. Electroejaculation is the most commonly used method in primates but is stressful for males because of the need for anesthesia [13]. Cauda epididymis is also reliable, but it is an unrepeatable and invasive method. Penile vibratory stimulation is a noninvasive repeatable method and yields a natural emission. Therefore, we developed a novel AI protocol using PVS.

In this study, we developed a modified AI technique using sperm collected by a method combining PVS and the sperm swim-up (SU) technique (hereafter, PVS–SU method). In addition, we also determined the optimal day(s) for AI by measuring plasma PRG during the ovarian cycle to determine when females should be treated with cloprostenol.

Section snippets

Animals

Marmosets from the Central Institute for Experimental Animals (Kawasaki, Japan) were used. The marmosets were reared in our facility under a 12/12-hour light/dark cycle at 24 ± 2 °C with 50 ± 5% humidity. Water and diet (CM-1; CLEA Japan, Tokyo, Japan) were available ad libitum. Seven adult nulliparous female marmosets (mean age, 3.6 ± 1.9 years; range, 1.5–5.7 years; mean body weight, 367.7 ± 37.8 g; range, 324–420 g) were used for the AI study. Female marmosets housed alone in individual

Success rate of the PVS method

A total of 113 attempts were performed in 27 males, and 94 ejaculate samples were obtained using the PVS method. The success rate was 83.2%. The hand-reared and virgin males yielded ejaculate, but two males did not provide semen after three attempts (Supplementary Table 1).

Artificial insemination

Sperm was prepared 21 times using 12 males and the PVS-SU method. Sperm concentration and motility were determined as shown in Table 1. Depending on the semen volume obtained by the PVS–SU method, sperm concentrations were

Discussion

We developed a modified AI method in which the SU method was combined with PVS to perform AI in marmosets. The SU technique has become a standard step to prepare sperm before assisted reproductive technology in humans [23]. The purpose of SU is to separate motile sperm from seminal plasma, dead sperm, leukocytes, and acellular debris [24]. Accordingly, highly concentrated motile sperm were obtained from 12 males using this method in this study. The SU method has been applied to human and NHP

Acknowledgments

The experimental protocol and design were approved by the Animal Experimentation Committee of Hamamatsu University School of Medicine and performed according to the Guidelines for Animal Experimentation.

The authors thank Kazuo Tanaka for his technical assistance.

References (28)

  • P.M. Summers et al.

    Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus)

    J Reprod Fertil

    (1985)
  • K.H. Cui et al.

    Collection and analysis of semen from the common marmoset (Callithrix jacchus)

    J Androl

    (1991)
  • C.H. Yeung et al.

    Maturation of sperm motility in the epididymis of the common marmoset (Callithrix jacchus) and the cynomolgus monkey (Macaca fascicularis)

    Int J Androl

    (1996)
  • I. Kuederling et al.

    Non-invasive collection of ejaculates from the common marmoset (Callithrix jacchus) using penile vibrostimulation

    Am J Primatol

    (2000)
  • Cited by (0)

    View full text