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