Behav Ecol Sociobiol (2007) 61:1759–1764 DOI 10.1007/s00265-007-0408-0 ORIGINAL PAPER Increased sperm numbers in the vas deferens of meadow voles, Microtus pennsylvanicus, in response to odors of conspecific males Javier delBarco-Trillo & Michael H. Ferkin Received: 26 September 2006 / Revised: 18 January 2007 / Accepted: 24 April 2007 / Published online: 9 May 2007 # Springer-Verlag 2007 Abstract Sperm competition occurs when the sperm of two or more males compete to fertilize the egg/s of a particular female. Males of some species respond to a high risk of sperm competition by increasing the number of sperm in their ejaculates. Males may accomplish such a response by increasing the intensity or duration of contraction of the cauda epididymidis and vas deferens. During emission (first phase of the ejaculatory process), the vas deferens receives sperm from the cauda epididymidis and propels the sperm to the urethra. In this paper, we tested the hypothesis that males exposed to a high risk of sperm competition mobilize larger numbers of sperm from the cauda epididymidis to the vas deferens before initiation of copulatory behavior. This accumulation of sperm in the vas deferens would result in a larger number of sperm in the ejaculate. To test this hypothesis, we exposed male meadow voles, Microtus pennsylvanicus, to either low or high risks of sperm competition using soiled bedding of conspecific individuals. At three different times after this exposure (15, 30, or 60 min), we removed both vasa deferentia and counted the sperm within them. We found a significant increase in sperm numbers in the vas deferens of males after 30 min of being exposed to a high risk of sperm competition. The lower sperm numbers after 15 and 60 min Communicated by E. Korpimäki J. delBarco-Trillo : M. H. Ferkin Department of Biology, University of Memphis, Ellington Hall, Memphis, TN 38152, USA J. delBarco-Trillo (*) Department of Psychology, Cornell University, Uris Hall, Ithaca, NY 14853, USA e-mail: jd333@cornell.edu of exposure suggest that the observed response is relatively slow and that sperm mobilized to the vasa deferentia may return to the cauda epididymides if ejaculation does not occur some time after the observed response. Our results indicate that the physiological response that may result in high sperm numbers in the ejaculate in relation to high risk of sperm competition can occur before initiation of copulatory behavior. Keywords Microtus . Rodents . Sperm competition . Sperm numbers . Vas deferens Introduction Sperm competition occurs when the sperm of two or more males compete to fertilize the egg/s of a particular female (Parker 1970; Birkhead and Møller 1998). Sperm competition has important effects on the morphology, physiology, and behavior of males (Dixson and Anderson 2004). Much research has been focused on the response of males to particular contexts of sperm competition (delBarco-Trillo and Ferkin 2004, 2006; Pound and Gage 2004). For example, a response of males to high risk of sperm competition may be to increase the number of sperm in their ejaculate (delBarco-Trillo and Ferkin 2004; Pound and Gage 2004). However, we do not know how males may process information about risk of sperm competition to generate such an increase in sperm numbers in the ejaculate (delBarco-Trillo and Ferkin 2005). Recently, we suggested that, when a male encounters information about high risk of sperm competition, such as when another male’s odors are in close proximity to his mate, that male displays a prompt physiological response that leads to an increase in sperm numbers in the ejaculate (delBarco-Trillo and Ferkin 2005). 1760 One such response a male may display when receiving information about high risk of sperm competition may involve an accumulation of sperm in the vas deferens. The vas deferens serves as a second reservoir of sperm, receiving sperm from the main reservoir, the cauda epididymidis. During emission (the first phase of the ejaculatory process), the vas deferens receives sperm from the cauda epididymidis and propels the sperm to the urethra. The vas deferens, however, is not just a tubule trough which sperm passes during ejaculation, but may also be an active organ during periods of sexual inactivity (Prins and Zaneveld 1979, 1980). Consequently, exposure to a high risk of sperm competition may trigger the mobilization of a high number of sperm from the cauda epididymidis to the vas deferens before copulatory behavior. By increasing the number of sperm in the vas deferens before copulation, a male may mobilize more sperm to the urethra during emission, which will result in a larger number of sperm in his ejaculate (Ratnasooriya and Wadsworth 1987). Some previous studies support this hypothesis. When comparing two related species of mice with differing levels of sperm competition, Pound (1999) found that the vas deferens of the promiscuous species was more sensitive to stimulation than the vas deferens of the monogamous species. It was concluded that the vas deferens of the species in which sperm competition is common may increase the number of sperm in the vas deferens in response to a high risk of sperm competition (Pound 1999). Another study compared the vasa deferentia of 103 species with different levels of sperm competition (Anderson et al. 2004). In those species in which sperm competition is common, males have shorter and more muscular vas deferens than in species in which sperm competition is rare, which may result in differential numbers of sperm in the ejaculate depending on different risks of sperm competition (Anderson et al. 2004). The previous studies suggest that the vas deferens may be an evolutionary malleable structure and that differences observed among species may be due to different levels of sperm competition. We hypothesize that males exposed to a high risk of sperm competition mobilize larger numbers of sperm from the cauda epididymidis to the vas deferens before initiation of copulatory behavior. We tested this hypothesis in meadow voles, Microtus pennsylvanicus. Male meadow voles make an excellent focal species for this study because high risk of sperm competition triggers an increase in sperm numbers in the male’s ejaculate but does not change their copulatory behavior (delBarco-Trillo and Ferkin 2004, 2007). Thus, an increase in sperm numbers in the ejaculate in response to high risk of sperm competition may be attributed to some type of physiological response and not to changes in copulatory behavior. To test the above hypothesis, we exposed male meadow voles to low and high risks of sperm competition and then Behav Ecol Sociobiol (2007) 61:1759–1764 measured the number of sperm in the vas deferens at different times after exposure. The low and high risks of sperm competition were characterized by exposing male meadow voles to combinations of bedding scented by male conspecifics, bedding scented by female conspecifics, or unscented bedding. Low risk of sperm competition involved bedding scented by females and unscented bedding, whereas high risk of sperm competition involved bedding scented by a conspecific male (delBarco-Trillo and Ferkin 2004, 2006). Materials and methods Animals The meadow vole is a promiscuous species for which multiple paternity has been shown to occur in the field (Boonstra et al. 1993). The importance of sperm competition has also been shown in laboratory conditions (delBarcoTrillo and Ferkin 2004, 2006, 2007). All meadow voles used in the study were second- and third-generation offspring of field-caught animals, born and raised in a temperaturecontrolled room with a 14:10 h light/dark cycle with lights on at 07:00 central standard time. This photoperiod simulates a day length typical of the breeding season. All tests were run during the first 2 h of the light cycle. Voles were weaned at 19 days of age, housed with littermates until 34 days of age, and then housed singly in clear polycarbonate cages (27 L×16.5 W×12.5 H centimeters). Cages contained hardwood shavings as bedding and cotton as nesting material. Food (Formulab Diet 5008, PMI Nutrition International, St. Louis, MO, USA) and water were provided ad libitum. We used sexually experienced male voles (average age=398±28 days of age). All odor donors were also sexually experienced. All focal males had not mated and had been isolated for 30 days before the start of the experiment. Isolation was threefold. First, males were kept in a room separated from the colony room. A maximum of nine males were kept in this isolation room. As males were tested and thus spaces became vacant, new males were brought to this isolation room. Second, cage tops with filters were placed over each cage to avoid any odors from entering into the cages. Third, all animals used in the study were visually isolated from each other by using wood partitions between cages. Groups There were three groups (C–C, FB–C, and FB– MB; see below), each one characterized by the odors that males were exposed to. We used three types of bedding containing different odors (delBarco-Trillo and Ferkin 2004). The control bedding (C) consisted of 25 g of clean bedding soaked in tap water. The female bedding (FB) consisted of 25 g of soiled bedding collected from the cage Behav Ecol Sociobiol (2007) 61:1759–1764 of a conspecific female. The male bedding (MB) consisted of 25 g of soiled bedding collected from the cage of a conspecific male. FB and MB were 4–5 days old and contained both urine and feces. Males in the C–C group were exposed to control bedding twice, with a 10 min interval between the two exposures. Males in the FB–C group were first exposed to female bedding and, after a 10-min interval, to control bedding. Males in the FB–MB group were first exposed to female bedding, and after a 10-min interval, they were exposed to male bedding. These three groups characterize different risks of sperm competition. In the C–C and FB–C groups, the risk of sperm competition is low, because the male is not exposed to the odors of any competing male (delBarco-Trillo and Ferkin 2004, 2006). In the FB-MB group, however, the focal male, after detecting the odors of a sexually receptive female, was exposed to the odors of another male, a context characterizing a high risk of sperm competition (delBarco-Trillo and Ferkin 2004). Experimental design Seven days before the trial, the male was transferred to a clean cage (44.5 L×24 W×14.5 H centimeters) with solid black walls. This cage had an opening (7 W×8 H centimeters) in one of the small sides. This opening was closed by means of a sliding door. At the onset of the test, this sliding door was removed, and a box (14 L×6 W×6.5 H centimeters) containing the first type of bedding (control bedding in the C–C group, or female bedding in the FB–C and FB–MB groups) was immediately attached to the cage. All the sides of the box were solid, except the top side which was transparent. If the male did not investigate the box within 1 min, that trial was discarded. The male was allowed to investigate the bedding in the box for 10 min. After those 10 min, the box was removed, and the opening closed again with the sliding door. Ten minutes later, the door was removed again and a second box was coupled to the cage. This second box contained control bedding (in the C–C and FB–C groups) or male bedding (in the FB–MB group). If the male did not investigate the second box within 1 min, that trial was discarded (11.1% of males tested were discarded because they did not investigate the first or the second box). After 10 min, the second box was removed, and the opening in the cage was immediately closed with the sliding door. After the removal of the second box, males were left undisturbed for 5, 20, or 50 min (see sample sizes below). After the appropriate undisturbed time interval, the male was taken from the cage and anesthetized immediately using isoflurane. Once anesthetized, the male was killed via cervical dislocation. The abdominal wall of the male was opened longitudinally. A forceps was applied at the junction between the cauda epididymidis and the vas deferens. The vas deferens in meadow voles is a distinct, 1761 thin and non-convoluted tubule approximately 2.5 cm long (Hamilton 1941). While holding the vas deferens with tweezers, we made a first cut at the junction between the vas deferens and the prostate gland and a second cut where the vas deferens was pinched by the forceps. This process was applied first to the left vas deferens and then to the right vas deferens. Both complete vasa deferentia were placed in a 1.5-ml centrifuge tube containing 1.5-ml distilled water; distilled water kills the sperm allowing sperm counting. Testes were also removed and weighted. We minced the two vasa deferentia in the microcentrifuge tube using small scissors. The contents in the microcentrifuge tube were then transferred to a manual grinder. To collect any remaining sperm in the microcentrifuge tube, we placed 1.5 ml additional distilled water in the microcentrifuge tube, agitated the contents using a micropipette and poured the solution in the manual grinder. We grinded the tissue until no tissue was visible to the eye. Such manual grinding liberated the sperm into the solution without breaking the sperm, as attested by the fact that nearly all the sperm we viewed under the microscope were complete. The solution containing the sperm was transferred from the manual grinder to a 9-ml glass container. The solution was gently shaken before taking each sperm sample to conduct sperm counts. We conducted four sperm counts using an improved Neubauer hemacytometer. The average of the four sperm counts was used to estimate the total number of sperm in both vasa deferentia. The repeatability of these four sperm counts was very high (r=0.96). The individual counting the sperm was blind to the type of bedding that the male was exposed. Statistical analyses Sample sizes for the C–C, FB–C, and FB–MB groups were 16, 12, and 17, respectively. The number of males tested 5 min after removal of the second box were 6, 4, and 6 (in the C–C, FB–C, and FB–MB groups, respectively); the number of males tested 20 min after removal of the second box were 5, 3, and 5 (in the C–C, FB–C, and FB–MB groups, respectively); and the number of males tested 50 min after removal of the second box were 5, 5, and 6 (in the C–C, FB–C, and FB–MB groups, respectively). Given that males were assigned randomly to the treatment groups, there were no differences among the treatment groups in body size (F2,42 =1.73, P=0.19), testes size (F2,42 =0.22, P=0.81), or age (F2,38 =1.04, P=0.36). We used the Kolmogorov–Smirnov test to determine if the assumption of normality was met. The two continuous variables considered in this study were normally distributed (testes size, Z=0.85, P=0.63; sperm numbers in vas deferens, Z=0.96 P=0.31). Unless otherwise specified, we used the general linear model (GLM) for all analyses. Because testes size was positively correlated with sperm counts in vas deferens (partial correlation, controlling for 1762 group; r=0.31, df=42, P=0.042), we used testes size as a covariate in all GLM analyses. All values are indicated as mean±1 SE. All statistical analyses were two tailed and performed using SPSS 12.0 for Windows. We considered differences to be significant when α<0.05. Behav Ecol Sociobiol (2007) 61:1759–1764 sperm counts in the vas deferens at different time intervals (F2,13 =5.07, P=0.024), with 16.35±4.34 million sperm 15 min after exposure, 36.16±6.69 million sperm 30 min after exposure, and 21.99±3.09 million sperm 60 min after exposure (Fig. 1). The only significant pairwise comparison existed when we compared the sperm counts of male voles 15 and 30 min after they were exposed to male bedding (Q= 4.27, P<0.05). Results We found a significant difference in sperm numbers among the three groups (F2,35 =5.6, P=0.008), but not among the three different time intervals (F2,35 =0.9, P=0.42) when considering both group and time variables in the same GLM analysis. The interaction between group and time interval (F4,35 =2.38, P=0.07) was not significant. The post hoc comparisons showed that sperm counts in the vas deferens between the C–C and FB–C groups were not significantly different (Tukey–Kramer test, Q=0.22, P> 0.05). In contrast, the sperm counts in the FB–MB group were significantly larger than in the C–C group (Q=3.53, P<0.05) and the FB–C group (Q=3.52, P<0.05). Thus, the number of sperm in the vas deferens is significantly larger among males that are exposed to bedding scented by another male. Sperm counts in the vas deferens did not change at different times after exposure in the C–C group (F2,12 = 0.11, P=0.89; average across times, 15.18±3.13 million sperm) or the FB–C group (F2,8 =2.19, P=0.17; average across times, 14.57±1.98 million sperm) when considering each group separately (Fig. 1). In the FB–MB group, however, there was a significant difference between the Fig. 1 Number of sperm (in millions) in the vas deferens after exposure to different risks of sperm competition. Males in groups C–C and FB–C were exposed to a low risk of sperm competition, whereas males in the FB–MB were exposed to a high risk of sperm competition. The number of sperm in the vas deferens of males in the FB–MB group was significantly larger than in the C–C and FB–C groups (F2,35 =5.6, P=0.008). Error bars indicate SE Discussion Ours is the first study to investigate whether the vas deferens modulates the number of sperm in the ejaculate of males exposed to different risks of sperm competition. We found that, when males are exposed to a high risk of sperm competition, they increase the number of sperm in the vas deferens. This increase in sperm numbers occurred in response to a high risk of sperm competition before any copulatory behavior. This finding suggests that a male may encounter information about risk of sperm competition and respond physiologically to that information so that, later on, when that male copulates with a female, he may ejaculate an appropriate number of sperm (delBarco-Trillo and Ferkin 2005). In the context of the present study, the information received by the male was the odors of a conspecific male soon after encountering the odors of a receptive female; such information indicated the presence of a competing male and, thus, a high risk of sperm competition. The physiological response may involve an increase in the contractility of the cauda epididymidis and vas deferens that results in an accumulation of sperm in the vas deferens and a subsequent increase in the number of sperm propelled to the urethra during ejaculation (delBarco-Trillo and Ferkin 2004). Although there is no reason to believe that the present results were biased by laboratory circumstances (e.g., Mappes et al. 1998), future research may consider the validity of our results in natural environments. The increase in sperm numbers in the ejaculate of male meadow voles is not trivial. Male meadow voles copulating in a context with a high risk of sperm competition ejaculate 116% more sperm within the female than do males copulating in a context with low risk of sperm competition (delBarco-Trillo and Ferkin 2004). The reported average increase in sperm numbers in the ejaculate in response to a high risk of sperm competition was 71 million sperm (delBarco-Trillo and Ferkin 2004). In the present study, the largest increase in sperm numbers in the vas deferens in response to a high risk of sperm competition was 26 million sperm. The increase in sperm investment reported by delBarco-Trillo and Ferkin (2004), therefore, cannot be explained only by the increase in sperm numbers in the vas deferens reported in this study. However, the results of this Behav Ecol Sociobiol (2007) 61:1759–1764 study suggest that the contraction of the cauda epididymidis and vas deferens is enhanced when a male is exposed to a high risk of sperm competition. Such an increase in contraction may mobilize more sperm not only before copulatory behavior but also during some of the ejaculations that compose copulatory behavior in this species (Pierce et al. 1990; delBarco-Trillo and Ferkin 2004). We found that, under a high risk of sperm competition, sperm numbers in the vas deferens were the highest 30 min after exposure to the odors of a conspecific male (Fig. 1). Conversely, sperm numbers in the vas deferens were much lower 15 and 60 min after exposure to the odors of a conspecific male. Low sperm numbers 15 min after exposure to the odors of a conspecific male suggests that the physiological response that triggers an increase in the muscular contraction of the cauda epididymidis and vas deferens may be relatively slow (see below). Low sperm numbers 60 min after exposure to the odors of a conspecific male suggest that sperm that were mobilized at the peak of sperm concentration in the vas deferens (30 min) may have returned to the cauda epididymidis. There is support for retrograde movement of sperm from the vas deferens to the cauda epididymidis in male rabbits (Prins and Zaneveld 1980). In that study, radio-opaque dye placed in the cauda epididymidis moved into the vas deferens after sexual stimulation (Prins and Zaneveld 1980). Fifteen minutes to 24 h after sexual stimulation without ejaculation, all the dye in the vas deferens had moved back to the cauda epididymidis, indicating that sperm transport can occur in both directions between the cauda epididymidis and the vas deferens (Prins and Zaneveld 1980). Similar results were obtained in male dogs (Kihara et al. 1995). The retrieval of sperm back to the cauda epididymidis some time after exposure to a high risk of sperm competition without the occurrence of mating is ecologically relevant in that if after a given time a male does not encounter fresh odors and scent marks of another male, the risk of sperm competition will decrease substantially (delBarco-Trillo and Ferkin 2005). Under such conditions, an enhanced activity of the cauda epididymidis and vas deferens may not be the best response (Dewsbury 1982). Although we did not test this in our study, it is possible that a repeated exposure to a high risk of sperm competition may result in a continuous high number of sperm in the vas deferens. A future line of research should investigate the pathway from reception of the odor characterizing a high risk of sperm competition to the increased mobilization of sperm from the cauda epididymides to the vasa deferentia. Although the chemical signals are received by the main olfactory receptors and/or vomeronasal organ (Brown 1985; Doty 1986), it is not clear which brain areas receive the incoming information from the olfactory system, although the medial amygdala and the medial preoptic area are 1763 potential candidates (Newman 1999; Fewell and Meredith 2002). Furthermore, it is not clear whether the processing of this information triggers a hormonal response, the production of an impulse through sympathetic efferent pathways directly to neuromuscular fibers in the epididymidis and vas deferens, or a combination of both (Kihara and De Groat 1997). In any case, the ultimate response is an increase in the intensity or duration of contraction of the developed musculature of the cauda epididymidis and vas deferens, resulting in a peristaltic pumping of sperm from the cauda epididymidis to the vas deferens (Baumgarten et al. 1971; Batra 1974). Some studies show a possible involvement of the hormone oxytocin in the peristaltic pumping of sperm from the cauda epididymidis to the vas deferens (Fjellström et al. 1968; Hib 1974; Knight 1974a, b; Sharma and Hays 1976; Arletti et al. 1985). There is also support for the production of an impulse that travels down the spinal cord from the brain to the nerves that innervate the muscles in the cauda epididymides and vasa deferentia (Cross and Glover 1958; Batra 1974; Kolbeck and Steers 1992; Ventura et al. 1973; Kihara and De Groat 1997). Contraction of the cauda epididymidis and vas deferens also occurred in response to electrical stimulation of the dorsal, lateral or posterior areas of the hypothalamus (Cross and Glover 1958). 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