This study has described an improved analysis of spiny mouse copulatory behaviour from that published by Dewsbury and Hodges  and provided new data on the relationship between mating success and the spiny mouse menstrual cycle in our captive colony. In their original study, Dewsbury and Hodges argued that male copulatory patterns cannot be predicted from knowledge of the female estrous cycle. Our study challenges this assertion. We have demonstrated the clear presence of a broad ‘mating window’ in which mating can occur during the early follicular, late follicular, and early luteal phase phases of the menstrual cycle, but not during the late luteal or menses phases. In mammals, gonadal steroids are known to affect sexual behaviour and mating receptivity . This relationship is true in female rodents , and also women, where copulation is significantly more frequent during their ovulatory window [16, 17] when testosterone and dihydrotestosterone (DHT) concentrations are the highest . Although androgen concentrations during the menstrual cycle in A. cahirinus have not been reported, our observations that females may also be receptive to mating several days either side of ovulation suggest a possible relationship between copulation frequency and changing androgen levels.
Unexpectedly, neither the pair-birth interval nor the number of pups born were significantly different across cycle phases where pups were born. This is in contrast to hamsters and mice where a significant difference in litter size is attributed to timing of mating [19, 20]. While time between the early follicular and early luteal phases of the menstrual cycle may differ by up to 2 days in the spiny mouse , it appears this time may be too little to observe a noticeable difference in litter size and pair-birth intervals. Further, a strong inverse relationship between litter size and gestation length has been reported in mice, rats, and gerbils [20,21,22]. However, differences are only apparent when comparing higher- and lower-order pregnancies in these species. Given the naturally small litter sizes in spiny mice (1–5; ) and higher-order pregnancies generally being born to multiparous dams (unpublished data from our colony), this may explain the similar pair-birth intervals we have observed.
Moreover, individual pairs established during the early follicular to early luteal phases of the cycle produce litters after a similar period to those reported in postpartum female spiny mice (41 days ± 4.4 days (SD); unpublished data from our colony). While litters were born > 45 days post-pairing in luteal phase females, these are most likely resultant of mating and pregnancies from the subsequent cycle, rather than the paired cycle. Thus, our method of timing pairing to a particular phase of the menstrual cycle, as outlined here, resulted in similar gestational outcomes to mated postpartum dams, while reducing study duration and costs.
Another distinction between our study and that of Dewsbury and Hodges , is the use of vaginal lavage to confirm behavioural cues for, and the timing and location of, ejaculation within the female reproductive tract. No spermatozoa were present in vaginal lavages taken immediately after individual, non-locking, intromission events. Despite interruptions to mating activities to obtain vaginal smears, and the potential stress involved in this procedure, spiny mice pairs rapidly resumed sexual activity after each consecutive vaginal lavage. This is an interesting and important observation for future mating studies because, although spiny mice are susceptible to stress (unpublished data from our colony), it appears that the mating drive is strong enough to overcome any stress caused by the disturbance of removing females briefly for vaginal lavage. Spermatozoa were seen in vaginal smears of females immediately after a copulatory lock, suggesting intravaginal ejaculation. However, as post-coital reproductive tracts of females were not examined in this study, we cannot rule out the possibility of intrauterine or intracervical insemination, as occurs in some other species like pigs and the camelids .
Our mating behavioural analysis reveals several similarities to Dewsbury and Hodges . We observed no pelvic thrusting during either intromission or immediately prior to ejaculation, and a series of intromissions always preceding ejaculation. We also observed no obvious lordosis in female spiny mice during intromission or ejaculation; a feature that is typical of murid copulation . Instead, we observed a distinct male foot twitch behaviour, which was not reported by Dewsbury and Hodges . Interestingly, a similar behaviour, ‘thumping’, was reported in Mongolian gerbils in which either individual taps its hind feet against the cage floor immediately following coitus . However, the cause of this behaviour is ambiguous as it presents in both sexes in other non-coital settings and has been considered a sign of stress . In contrast, the male spiny mouse foot twitch that occurred only during coitus, we interpret as a behavioural response of males to pre-ejaculatory penile insertion during mounting on a female not presenting any clear lordosis. While multiple intromissions have been suggested as a necessary requirement to trigger ovulation in several rodent species [1, 27], A. cahirinus present with spontaneous, rather than induced ovulation [6, 7]. Therefore, the multiple intromissions observed here are more likely a prerequisite to stimulate ejaculation, rather than to stimulate ovulation in A. cahirinus.
We also confirm the presence of a copulatory lock and no obvious copulatory plug in A. cahirinus. In an extensive review of 118 mammalian species, Dewsbury  categorised mating behaviour into 16 categories; the most common patterns being # 9 (no lock, intravaginal thrusting, multiple intromissions and ejaculations) and #13 (no lock, no intravaginal thrusting, multiple intromissions and ejaculations). Interestingly, all species in these two categories were either primates or rodents, with most rodents falling into category #13. From this, it appears that mating behaviour in A. cahirinus is broadly similar to other rodent species but with the addition of a copulatory lock. Seminal plugs are common in rats and guinea-pigs, but only observed in a few mouse species , and these non-plugging species generally have copulatory locks and reduced or underdeveloped accessory glands (reviewed by ). Further, Voss  argued that if there is a causal relationship between the presence of copulatory locks and the absence of plug formation, they ‘must serve much the same function(s) as the plugs they presumably replaced’.
Male spiny mice have a normal complement of accessory glands typical of many murid rodents , with a large well-developed seminal vesicle but a comparatively small prostate and coagulating glands. Hartung and Dewsbury  have suggested that well-developed accessory glands are required for rodent seminal plug formation, but no copulatory plug has been observed in spiny mice. However, coagulation studies using spiny mouse accessory gland secretions , especially mixing of extracts from the seminal vesicles and the coagulating glands, shows coagulum formation. Together, this suggests the possibility of a covert post-ejaculatory seminal plug in spiny mice, perhaps deep within the vagina against the cervix or within the cervical canal.
The functional role of copulatory plugs in rodents has been debated for centuries  and several hypothesis have been suggested. These include prevention of insemination by rival males, assisting sperm transport, induction of pseudopregnancy and prevention of sperm leakage from the vagina . None of these hypothesis are likely to apply in A. cahirinus considering the extremely brief lock compared to true locking species , and the demonstrated inability to induce pseudopregnancy in this species . An alternative explanation is that, despite the brief ejaculatory lock, spiny mice deposit most or all of the ejaculate directly into the cervix or uterus like camelids and pigs . Although spermatozoa were seen in vaginal lavages immediately following the brief locking events, these spermatozoa may be flowback through the cervix or leakage from the penis during withdrawal from the vagina. Considering this, if ejaculation does occur within the cervix or the uterus, formation of a small, very temporary, seminal plug may assist in maintaining spermatozoa at the site of ejaculation. Future studies of female spiny mouse reproductive tracts following coitus may provide answers to these questions on the site of insemination and presence of post-coital seminal plug, and provide new information on sperm concentration, survival, and transit through the female tract.