Mating in the cold. Prolonged sperm storage provides opportunities for forced copulation by male bats during winter

Abstract

In a wide range of heterothermic mammals, hibernation interrupts the reproductive cycle by forcing reproductive delays. In hibernating bats with delayed fertilization, an opportunity for sperm competition is enhanced by extending a time-window between copulations and fertilization. In order to achieve greater fertilization success, males are expected to show adaptations for sperm competition by increasing their opportunities for mating over an extended period. We aimed to clarify the physiological and behavioral characteristics of male bats experiencing increased risks of sperm competition. We investigated the characteristics of the reproductive cycle of the little horseshoe bat (Rhinolophus cornutus), and examined whether males retain reproductive physiology related to sexual behavior, and attempt to copulate with females even during the hibernation period. Field observations and histological examinations of the reproductive cycle confirmed that females, having mated in the autumn, store spermatozoa in the uterus during hibernation and give birth in the early summer to just one offspring per year, thus males face a low certainty of successful fertilization. Although their testes regressed rapidly and their testosterone levels were lower during winter than in autumn, males stored motile spermatozoa in their cauda epididymides from autumn throughout the winter. During hibernation, we found that males occasionally aroused from torpor and attempted to mate forcibly with torpid females. Forced copulations appear to increase a male's chances of obtaining a mate while avoiding pre-copulatory female choice. Epididymal sperm storage could be advantageous for males in allowing them to extend their potential mating period even though their testes have regressed. We also found that some hibernating nulliparous females were ready for fertilization in spring after hibernation, whereas few parous females appeared in the same roost. In contrast to males, forced copulations would be maladaptive for females because they cannot opt for higher-quality males while in torpor. Females that have experienced sexual coercion when young may subsequently avoid hibernacula where adult males are present.

Keywords: Chiroptera; heterothermy; reproductive delay; sexual coercion; sperm competition.

FIGURE 1

Annual reproductive cycle of the little horseshoe bat (Rhinolophus cornutus) in Niigata Prefecture. (A) denotes reproductive cycle of both sexes determined by monitoring of reproductive conditions and histological examination of females in (B–D). Arrow heads represent the period of capture for female histological study. The beginning of spermatogenesis was not determined. (B–D) show ovary and uterus of adult females collected during post-lactation, early hibernation and late hibernation period. Arrow heads in (C,D) indicate stored spermatozoa found in the vicinity of the utero-tubal junction. Scale bars on HE stained images are 100 μm.

FIGURE 2

Sperm storage in cauda epididymis during hibernation. (A) represents seasonal changes in external testicular (solid line) and cauda epididymis size (dashed line). Measurements are shown as means ± SE. Months without symbols indicate that no males were captured. Light-gray bars represent hibernation. (B–D) show seasonal changes in external and internal morphology of gonads, and seminiferous tubules of testis and lumen of cauda epididymis. The arrow and arrow heads in (B,C) indicate enlarged testis and cauda epididymis, respectively. Scale bars on HE stained images are 100 μm.

FIGURE 3

Seasonal changes in male reproductive physiology. (A–C) show the weight of testis, cauda epididymis and complex of accessory sex glands during anestrus (n = 8), estrus (n = 10) and hibernation (n = 13). (D,E) represent seasonal differences in testosterone concentrations (n = 6 for both seasons) and percentages of motile spermatozoa (n = 3 in estrus, n = 4 in hibernation), respectively. Data are shown using boxplots; the top and bottom of the boxes represent the 75th and 25th percentile, respectively, and lines indicate the median. *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, ***: p < 0.001, n.s.: not significant. Statistics for (E) are shown in text and Supplementary Table S2.

FIGURE 4

Winter forced copulation by males periodically aroused from torpor. (A) is a representative image of a copulating pair. (B) shows thermal infrared image corresponding to the pair shown in (A), and approximate outlines (white lines) of bats. (C) indicates ejaculated semen (dashed circle) found in the vaginal opening of a post-copulatory female. (D) represents spermatozoa (arrow head) collected by vaginal smear.

FIGURE 5

Reproductive conditions of hibernating females. (A–C) represent adult parous, fertile nulliparous subadults, and non-reproductive females, respectively. Black arrows in the image of vagina (A) indicate pubic nipples (sign of lactating experience). Images of plugs show the cross sections of the vagina after preservation in 10% formalin neutral buffer solution, and the extracted plug. All scale bars on these images are 100 μm. (D) shows the comparison in percentages of histologically determined reproductive categories between females hibernating in a group and in those hibernating solitarily (see text for statistics). *: p < 0.05 (Fisher’s exact test).