Superconception in mammalian pregnancy can be detected and increases reproductive output per breeding season

ARTICLE Received 1 Feb 2010 | Accepted 24 Aug 2010 | Published 21 Sep 2010 DOI: 10.1038/ncomms1079 Superconception in mammalian pregnancy can be detected and increases reproductive output per breeding season Kathleen Roellig1, Frank Goeritz1, Joerns Fickel1, Robert Hermes1, Heribert Hofer1 & Thomas B. Hildebrandt1 The concept of superfetation, a second conception during pregnancy, has been controversial for a long time. In this paper we use an experimental approach to demonstrate that female European brown hares (Lepus europaeus) frequently develop a second pregnancy while already pregnant and thereby increase their reproductive success. After a new, successful copulation, we confirmed additional ovulations before parturition in living, late-pregnant females by detecting a second set of fresh corpora lutea using high-resolution ultrasonography. The presence of early embryonic stages in the oviduct, demonstrated by oviduct flushing, next to fully developed fetuses in the uterus is best explained by passage of semen through the late-pregnant uterus; this was confirmed by paternity analysis using microsatellite profiling. Subsequent implantation occurred after parturition. This superfetation, categorized as superconception, significantly increased litter size and permitted females to produce up to 35.4% more offspring per breeding season. It is therefore most likely an evolutionary adaptation. Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, Berlin 10315, Germany. Correspondence and requests for materials should be addressed to K.R. (emails: or ). 1 nature communications | 1:78 | DOI: 10.1038/ncomms1079 | www.nature.com/naturecommunications © 2010 Macmillan Publishers Limited. All rights reserved.  ARTICLE nature communications | DOI: 10.1038/ncomms1079 H ow could a female become pregnant while she is still pregnant? And what is the benefit? In mammals, maternal provisioning includes the periods of intrauterine development and lactation. In short-lived mammals with early maturity and a short reproductive lifetime (often described as r-selected species)1, the number of reproductive cycles within a breeding period is usually considered to be maximized if the next fertilization follows immediately after parturition. In a polyoestrous seasonal breeder such as the European brown hare (Lepus europaeus), reproductive cycle length could be even further shortened if a new conception already occurred during an ongoing intact pregnancy, thereby increasing the potential reproductive output within a breeding period. A new conception during an intact pregnancy is defined as superfetation2 and was first suggested for the hare by Aristotle3. For superfetation to be successful, there are several challenges to overcome, such as stimulation of ovulation during pregnancy, which should be physiologically suppressed4, passage of semen through an already occupied uterus, considered to be unfeasible and unsuccessful5,6, and possibly implantation of a second set of embryos next to already implanted fetuses in the uterus. The diagnosis of superfetation in any species therefore requires an experimental demonstration of ovulation and fertilization during an intact pregnancy, the presence of discordantly developed offspring within the reproductive tract of the mother under tightly controlled conditions and the tracking of the two pregnancies irrespective of the mechanism that permits the phenomenon to occur2. As this has always been difficult for methodological reasons, superfetation has been controversial since ancient times2,3. Historically, the presence of discordantly developed fetuses within a uterus3,7 was usually discarded as evidence2, as other explanations were readily available2 and the physiological requirements for superfetation unlikely to be met. The European brown hare is a strictly induced ovulator; stimulation by copulation is considered a necessity7. In this paper, we investigate whether successful prepartum conception in European brown hares really exists, how possible mechanisms might operate and how such superfetation affects female reproductive success. Using a captive colony and a stepwise experimental approach, we demonstrate that superfetation in European brown hares exists, can be diagnosed in living individuals, is a widespread, regular occurrence, requires a second copulation during the last phase of the ongoing pregnancy and is most likely achieved by passage of semen through the latepregnant uterus. On the basis of our observations that superfetation increases litter size and reduces each reproductive cycle by 4 days, we discuss its evolutionary origin and conclude that superfetation in the European brown hare is an evolutionary adaptation to shift maternal care from the postpartum to the prepartum stage and thus move postpartum care towards a precocial mode. Results Detection of superfetation through breeding-pair management. In a large-scale experiment in a captive population of European brown hares, we studied pregnant females over four consecutive breeding seasons (January to September). In three groups of females, different tactics of breeding management were used. Copulation activity was confirmed by video surveillance. Mean pregnancy lasted 41.9 ± 0.7 days (n = 24 females and 35 litters), as measured in breeding pairs in which the male was only present for 1 day to procreate the initial litter and then removed (group A ‘conventional breeders’). In breeding pairs permanently housed together (group B ‘permanent breeders’), copulations were frequently observed by video surveillance 3–4 days before parturition. Parturition of successive litters occurred after an interval of 38.4 ± 1.0 days (n = 6 females and 18 litters)2,8,9. To exclude the possibility that group B males had escaped video surveillance and copulated after parturition and that gestation period in group B females was unusually variable, a third group of breeding pairs (group C ‘temporary breeders’) were  housed together temporarily for (1) 1 initial day to allow copulation, and after pregnancy was confirmed (2) for 5 days from day 36 to 41 of gestation. The interbirth interval in temporary breeders was 38.1 ± 1.8 days (n = 7 females and 11 litters). If the conception of a new litter only starts after parturition of the previous litter, the interval between successive litters (permanent and temporary breeders) should have the same duration as ‘normal’ pregnancies (conventional breeders). However, the observed differences between mean interbirth intervals of these groups were significant (Kruskal– Wallis test, H = 30.614, d.f. = 3 including group D, see below, P < 0.00001): the gestation period of conventional breeders was significantly longer than the interbirth interval of either permanent (post hoc multiple comparisons, t = 6.258, P < 0.00001) or temporary breeders (t = 6.636, P < 0.00001), whereas there was no difference between permanent and temporary breeder (...truncated)