Repeated Sampling Affects Tree Swallow Semen Characteristics/(Efecto de la repetición en la toma de muestras en las características del semen de Tachycineta …

Repeated sampling affects Tree Swallow semen characteristics Author(s) :M. P. Lombardo, M. L. Green, P. A. Thorpe, M. R. Czarnowski, and H. W. Power Source: Journal of Field Ornithology, 75(4):394-403. 2004. Published By: Association of Field Ornithologists DOI: 10.1648/0273-8570(2004)075[0394:RSATSS]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.1648/0273-8570%282004%29075%5B0394%3ARSATSS %5D2.0.CO%3B2

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J. Field Ornithol. 75(4):394–403, 2004

Repeated sampling affects Tree Swallow semen characteristics M. P. Lombardo,1,4 M. L. Green,1 P. A. Thorpe,1 M. R. Czarnowski,2 and H. W. Power3 2

1 Department of Biology, Grand Valley State University, Allendale, Michigan 49401 USA Graduate Program of Ecology and Evolution, Rutgers University, New Brunswick, New Jersey 08901 USA 3 Department of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, New Jersey 08901 USA

Received 29 October 2003; accepted 4 March 2004 ABSTRACT. Male Tree Swallows (Tachycineta bicolor) face intense sperm competition because mated pairs copulate frequently, extra-pair copulations are common, and females store sperm. We examined the effects of repeated sampling on the characteristics of Tree Swallow semen by manually expressing semen from 15 males immediately after capture (T0) and then hourly for 4 h (T1–T4). The semen characteristics of individual males varied in response to repeated sampling. The total number of sperm cells we obtained from each male over the 4h sampling period varied from 104–107. Semen samples lacking sperm increased from 6.7% of T0 samples to 26.7– 33.3% of subsequent samples. Forty percent of males provided at least one semen sample that lacked sperm. There were no significant differences among hourly samples in semen volume, sperm concentration, or in the total number of sperm cells obtained from each male. However, there were significant differences among males in each of these variables. Semen volumes represented small proportions of cloacal protuberance volumes. We did not detect significant correlations between total semen volumes or total number of sperm cells obtained from males from T0–T4 and cloacal protuberance volumes. Total semen volume and number of sperm cells obtained from T0–T4 significantly increased with date. However, sperm concentration was not significantly correlated with date. We did not detect significant correlations between semen characteristics and male morphology. Individual variation in responses to repeated sampling has implications for the copulatory strategies of male and female Tree Swallows. SINOPSIS.

Efecto de la repeticio´n en la toma de muestras en las caracterı´sticas del semen de Tachy-

cineta bicolor

Los machos de la golondrina Tachycineta bicolor, enfrentan una intensa competencia entre espermatozoides dado el caso de que las parejas copulan frecuentemente y que la copulacio´n, entre individuos que no son parejas, son comunes y que las hembras almacenan semen. Examinamos el efecto de sacar varias muestras de semen desde la captura del animal (T0) y luego cada hora por un lapso de cuatro horas (T1–T4) en las caracterı´sticas del semen de 15 individuos. Las caracterı´sticas del semen vario en respuesta al muestreo repetitivo. El nu´mero total de espermas que obtuvimos de cada macho en las cuatro horas de muestreo vario´ de 104 a 107. El nu´mero de muestras de semen sin espermatozoides vario desde 6.7% en el T0 hasta 26.7–33.3% en los muestreos subsiguientes. El 40% de los machos produjeron al menos una muestra sin espermatozoides. No se encontro´ diferencia significativa entre las muestras tomadas a cada hora, el volumen de semen, concentracio´n de espermatozoides, o en el nu´mero total de espermatozoides obtenidos de cada macho. Sin embargo, hubo diferencia significativa entre machos individuales en cada una de las variables. El volumen de semen represento´ una proporcio´n pequen˜a del volumen de la protuberancia cloacal. No detectamos correlacio´n significativa entre el volumen total de semen o el nu´mero total de espermatozoides obtenidos de machos del T0 al T4 y el volumen de la protuberancia cloacal. El volumen total de semen y el nu´mero de espermatozoides obtenidos desde el T0 al T4 aumento significativamente con la fecha de colecta. Sin embargo, la concentracio´n de semen no correlaciono´ significativamente con las fechas. No detectamos correlacio´n significativa entre las caracterı´sticas del semen y la morfologı´a del macho. La variacio´n individual en respuesta a la toma repetida de muestras tiene implicaciones importantes en lo que respecta a las estrategias copulatrices en machos y hembras de esta especeie de golondrina. Key words: extra-pair copulations, fertility insurance, sperm competition, sperm depletion, sperm reserves, Tachycineta bicolor, Tree Swallows

In many species of birds, sperm cells from two or more males may compete (Parker 1970) 4

edu

Corresponding author. Email: lombardm@gvsu.

to fertilize the eggs within a single clutch of eggs because the female participated in extrapair copulations (EPC) (Westneat et al. 1990; Birkhead and Møller 1992, 1998). Elucidating the dynamics of sperm competition is funda-

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mental to understanding sexual selection because the consequences of sperm competition affect the evolution of male behavior and physiological traits that maximize paternity (Birkhead and Møller 1992, 1998). In species with intense sperm competition, selection should favor males that are able to inseminate females with large numbers of sperm cells (Parker 1970; Møller 1988; Birkhead et al. 1993). Consistent with this prediction, testis size, sperm counts, and ejaculate size are positively correlated with the probability of sperm competition in some species (e.g., Castro et al. 1996; Tuttle et al. 1996; Sax and Hoi 1998; Brown and Brown 2003). The outcome of sperm competition in birds may be influenced by the interaction of many factors (Birkhead and Møller 1992, 1998; Birkhead et al. 1995; Eberhard 1996; Colegrave et al. 1995; Birkhead and Biggens 1998; Michl 2002; Skau and Folstad 2003), including the sperm concentration of ejaculates (Martin et al. 1974). Sperm concentration in birds may be influenced by a combination of factors including intrinsic differences among males (Lombardo et al. 2002), the risk of sperm competition (Nicholls et al. 2001; Pizzari et al. 2003), seasonal patterns of sperm production (Lombardo et al. 2002), and copulation frequency (Birkhead 1991; Birkhead and Fletcher 1995; Birkhead et al. 1995; Kast et al. 1998; Westneat et al. 1998). Changes in semen characteristics influenced by copulation frequency are likely to have important effects on male reproductive success in species characterized by frequent copulations. Our goal was to examine the effects of repeated sampling, designed to mimc repeated bouts of copulation, on Tree Swallow semen characteristics. Male Tree Swallows are excellent candidates for studying the effects of repeated sampling on semen characteristics for several reasons. First, mated Tree Swallows copulate frequently (ca. 51 times per clutch of 4–7 eggs; Venier and Robertson 1991; Robertson et al. 1992). Second, EPCs (Lombardo 1986; Venier et al. 1993) and extra-pair paternity (e.g., 50– 87% of broods have extra-pair paternity; Dunn et al. 1994; Barber et al. 1996; Whittingham and Dunn 2001) are common. Chek and Robertson (1994) argued that male Tree Swallows attempt to avoid being cuckolded not by mateguarding (Leffelaar and Robertson 1984), but

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with frequent within-pair copulations. Frequent inseminations can improve a male’s chances of fertilizing eggs (Birkhead and Møller 1992; Hunter et al. 1992; Westneat 1993). This hypothesis predicts that male Tree Swallows should produce large numbers of sperm cells to avoid sperm depletion. Last, the high frequencies of within- and extra-pair copulations, and the lack of close mate-guarding, together with the presence of sperm storage tubules in females (Shugart 1988) favor a high rate of sperm production (Birkhead et al. 1993). And consistent with this prediction, cloacal protuberance (CP) dimensions (Lombardo 2001), testes volume (Peer et al. 2000), sperm storage tubule number and length, and the length of Tree Swallow sperm cells are more similar to those of some polygynandrous and polygynous bird species (in which males typically encounter intense sperm competition) than those of other socially monogamous birds (Briskie 1993). METHODS

We collected semen from 15 male Tree Swallows that bred in wooden nest boxes arranged in grids in an old field on the campus of Grand Valley State University (428579N, 858539W), Ottawa County, Michigan, USA, in 2000. We began daily monitoring of nest boxes in late April. Female swallows laid eggs from early May to mid-June. We checked nests daily during egg laying and then periodically afterwards to keep track of incubation, hatching, and fledging. We captured swallows at their nest boxes between 07:00 and 12:00 EDT during incubation periods at each nest. We distinguished males by noting the presence of a CP, the lack of a brood patch, and plumage characteristics (Cohen 1980, 1984; Hussell 1983; Stutchbury and Robertson 1987; Peer et al. 2000). Each swallow was banded with a U.S. Fish and Wildlife Service numbered aluminum band and its plumage marked with a unique color-mark using a waterproof marker to facilitate individual identification after they were released. We measured each male’s (1) right tarsus length with an electronic digital caliper to 0.01 mm, (2) unflattened right wing chord length to 1 mm with a ruler with a stop fixed to one end, and (3) mass to the nearest 0.2 g with an Avinet spring scale. One wing with frayed feathers was not measured. Body condition was estimated as

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mass divided by right tarsus length. Tree Swallows have slightly forked tails (Robertson et al. 1992) and are sexually dimorphic for this trait (M. Lombardo, unpubl. data). The degree of tail forking was determined by measuring the length of tail feathers from the notch in the center of the slightly forked tail to the tip of the outer tail feathers on the right side of the tail to the nearest 1 mm with a ruler while the tail was held so that the outer edges of each outer tail feather were parallel to each other. We collected semen samples from males while their mates were incubating because we could more reliably obtain semen samples from males during incubation than at any other period in the nesting cycle (Lombardo et al. 2002). Within-pair copulations are rare while females are incubating eggs (Venier and Robertson 1991; Robertson et al. 1992). Female Tree Swallows typically incubate eggs for 14 d (Robertson et al. 1992). We collected samples on incubation days 10 (N 5 11), 11 (N 5 2), 13 (N 5 1) and 14 (N 5 1). Two males were sampled on 20 May, two on 21 May, and single males were sampled each day on 22–25, 29–31 May and 2–4, 6 June. Obtaining samples during the latter half of incubation probably had little influence on semen quality because Tree Swallow sperm concentrations are not significantly correlated with date during incubation periods (Lombardo et al. 2002). Males were sampled immediately after being captured (T0) and then hourly for 4 h (T1–T4). This sampling regime was chosen to mimic multiple bouts of copulation at 1-h intervals over the course of a morning. Tree Swallow males average approximately 0.5 copulation attempts/h with their mates during the morning (Venier and Robertson 1991), but multiple within-pair copulations occurring at intervals shorter than 1 h are not uncommon (M. Lombardo, pers. obs.). Each successful copulation bout consists of 1–18 cloacal contacts (Petersen et al. 2001). It is not known if semen is transmitted during all cloacal contacts. Males were held in vacant nest boxes with window screen fitted over the hole so they could be re-sampled hourly without having to be recaptured. We do not think that holding males for 4 h in a vacant nest box without access to food or water had an adverse effect on the size of their sperm reserves during our sampling because it is likely that their daily sperm reserves were produced

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before we captured them (Riley 1937; Middleton 1974; Quay 1987). None of the sampled males abandoned their nests after being released from detention. We manually expressed semen from males by applying gentle pressure to the seminal glomera in the CP (Samour et al. 1986). Lombardo collected all semen samples. During each sample period we collected semen samples in 0.5 ml volumes with sterile micropipets until no more semen could be expressed from the cloaca. During some sample periods we were unable to obtain semen samples from some males and only trace amounts from others (see Results). Trace amounts were defined as samples that were less than 0.5 ml and were recorded as 0.1 mL. Samples were immediately transferred to sterile containers containing 100 ml of 5% buffered formalin and placed on ice for transport to the laboratory. The samples were thoroughly mixed in the laboratory by vortexing on a Thermolyne MAXIMIN PLUSy (Barnstead/Thermolyne, Dubuque, Iowa) for 10 s, then 50 ml was transferred into 500 ml of 5% buffered formalin for sperm counting. The remaining volume was used for plating for microbes (e.g., Lombardo and Thorpe 2000). Samples were stored at 48C until counted by MLG. Three sub-samples from each stored sample were counted using an improved Neubauer hemacytometer (Reichert, Buffalo, New York) at 10003 magnification on a compound microscope. We used the mean sperm concentration (sperm/ml) of the sub-sample counts to calculate the total number of sperm cells obtained from each male. Tree Swallow males store sperm in the seminal glomera of the CP (Peer et al. 2000). Larger CPs have been hypothesized to be adaptations for storing larger sperm reserves (Birkhead et al. 1993). Evidence from Australian fairywrens (Malurus spp.) supports this hypothesis (Tuttle et al. 1996). We measured CP dimensions and calculated CP volume following Lombardo (2001), to determine if CP volume accurately reflected the size of obtainable sperm reserves in Tree Swallows. Semen characteristics may be influenced by collection technique (Gee and Temple 1978). Cloacal massage may not be a reliable method for estimating natural ejaculate size (Pellatt and Birkhead 1994). However, our goal was to determine whether repeated sampling affected se-

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men characteristics, not to estimate ejaculate size. Tuttle et al. (1996) used cloacal massage to obtain semen to examine semen characteristics in Australian Fairy-Wrens (Malurus spp.). We estimated the number of fertile females at the study site each day that we collected samples. The number of fertile females at the study site on each sampling day can be used as an upper estimate of the number of potential reproductive opportunities for males. A female was categorized as fertile from six days before she laid her first egg until the day she laid her penultimate egg (Venier and Robertson 1991). We used SPSS 8.0 for Windows (SPSS 1997) to examine the data for normality and used parametric and nonparametric statistical tests to analyze data where appropriate. We used the Gtest and Fisher exact test to determine if our ability to obtain semen from males was independent of sample period or if a sample containing sperm cells was independent of sample period, respectively. We used Pearson correlations to detect statistically significant correlations between semen sample characteristics (i.e., volume, sperm concentration, total sperm number), male physical characteristics, sample date, and the number of fertile females at the study site on sample date. We used repeated measures ANOVA to examine variation in semen characteristics across sample periods from individual males and among males related to sample period. Sequential Bonferroni corrections were used to detect statistically significant relationships in multiple comparisons (Rice 1989). Only analyses where sequential Bonferroni corrections altered our interpretations are reported. Data are reported as means 6 SD unless stated otherwise. RESULTS

We obtained multiple semen samples from 15 males. The volume of semen we obtained at T0 was significantly correlated with that obtained at T1 (r 5 0.94, P , 0.001, N 5 15), indicating that the semen volume obtained from a male during any sampling attempt was not due to differences in our sampling efforts across sample periods. Our ability to obtain a measurable semen sample was independent of the sample period (T0–T4) from which it was obtained (G4 5 4.19, P . 0.25). Whether sperm was present

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Fig. 1. The proportion of Tree Swallow semen samples during which we either did not obtain semen or the semen sample was devoid of sperm increased from 6.7% at T0 to between 26.7–33.3% over the rest of the sampling period.

in a sample or not was independent of whether it was the first or subsequent sample (Fisher exact test, P 5 0.17, Fig. 1). However, the percentage of samples lacking sperm increased from 6.7% at T0 to 26.7–33.3% during subsequent sampling periods (Fig. 1). Forty-four of 60 (73%) T1–T4 samples contained sperm. We obtained samples containing sperm from nine of 15 (60%) males during all five sampling attempts. In contrast, six of 15 (40%) males provided at least one sample lacking sperm. We did not detect any significant relationships between sampling attempt and mean semen volume (ANOVA, F4 5 1.70, P 5 0.21), mean sperm concentration (F4 5 2.42, P 5 0.07), or mean number of sperm cells obtained (F4 5 3.01, P 5 0.08) from individual males (Table 1). We used sperm concentration at T0 to examine the relationship between sperm concentration and sampling date because there was no significant difference between sampling attempts from T0–T4 in mean sperm concentration. Sperm concentration did not significantly vary with sampling date (r 5 0.23, P 5 0.43, N 5 15). There was significant variation among males. Individual males varied by orders of magnitude (104–107) in the total number of sperm cells we obtained from them from T0– T4 (repeated-measures ANOVA, F1,13 5 12.90, P 5 0.003), mean sperm concentration (F1,8 5 53.55, P , 0.001), and the mean number of sperm cells (F1,8 5 16.24, P 5 0.004) we obtained from them during each sampling attempt from T0–T4 (Table 1). Across all males, the mean number of sperm cells we obtained during each sample period

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Table 1. The characteristics of semen obtained from male Tree Swallows by cloacal massage were affected when sampled immediately after being captured (T0) and then hourly (T1–T4) for four hours. Mean 6 SD (sample size) are shown. Sample period T0 T1 T2 T3 T4

Semen volume (mL) 4.40 2.60 3.03 1.34 2.17

6 6 6 6 6

5.77 3.72 4.61 1.49 3.97

(15) (15) (15) (15) (15)

Sperm concentration (106 sperm/mL) 5.22 6.34 4.30 3.73 4.40

significantly differed (repeated measures ANOVA, F4 5 5.59, P 5 0.04; Table 1). The mean number of sperm cells we obtained from the nine males from whom we obtained five semen samples differed significantly (repeated-measures ANOVA, F1,8 5 16.24, P 5 0.004). The number of fertile females present at the study site significantly declined (r 5 20.95, P , 0.001, N 5 15) from 20 May–6 June, as did the total volume of semen we obtained from each male (r 5 20.63, P 5 0.01, N 5 15). However, total semen volume was not significantly correlated with the number of fertile females at the study site (r 5 0.39, P 5 0.17, N 5 12) when sampling date was controlled for using partial correlation analysis. Likewise, the total number of sperm cells obtained from males at T0–T4 was not significantly correlated with the number of fertile females at the study site (r 5 0.09, P 5 0.77, N 5 12) when date was controlled for using partial correlation analysis. However, the total number of sperm cells we obtained from individual males significantly increased with sampling date (r 5 0.55, P 5 0.03, N 5 15). We did not detect significant correlations between the number of samples containing sperm we obtained from individual males and sampling date (r 5 0.04, P 5 0.14), mass (r 5 0.39, P 5 0.15), right tarsus length (r 5 0.37, P 5 0.18), right tail fork length (r 5 0.09, P 5 0.76), or body condition (r 5 0.14, P 5 0.62). Males with longer right wing chords produced more samples containing sperm (r 5 0.55, P 5 0.04, N 5 14), but after a sequential Bonferroni correction for multiple comparisons (critical value, P 5 0.008) this relationship was not significant. There were no significant differences between males from whom we obtained either five (N 5 9) or fewer than five (N 5 6) samples containing sperm from T0–T4, in

6 6 6 6 6

4.22 3.16 3.58 3.73 3.05

(14) (11) (11) (11) (11)

Sperm cells (106) 28.86 21.91 19.85 8.28 12.12

6 6 6 6 6

33.63 23.10 33.97 16.39 13.93

(14) (11) (11) (11) (11)

mass (t13 5 1.87, P 5 0.08), right wing chord length (t13 5 1.88, P 5 0.08), right tail fork length (t13 5 0.34, P 5 0.74), or body condition (t13 5 0.32, P 5 0.76). However, males from whom we obtained five samples containing sperm had significantly longer right tarsi (t13 5 2.39, P 5 0.03) than did males that produced fewer than five samples containing sperm. However, after a sequential Bonferroni correction for multiple comparisons (critical value, P 5 0.01), this relationship was not significant. We did not detect significant correlations between the total number of sperm cells obtained from males and their mass (r 5 0.48, P 5 0.07), right tarsus length (r 5 0.36, P 5 0.19), right wing chord length (r 5 0.21, P 5 0.48), right tail fork length (r 5 0.37, P 5 0.17), or body condition (r 5 0.23, P 5 0.40). Cloacal protuberance volume has been considered as a proxy for the volume of sperm reserves (e.g., Tuttle et al. 1996) because passerines store sperm in the seminal glomera within the CP. Tree Swallow CP volume significantly declines as the season progresses (Lombardo 2001). When sample date was controlled for using a partial correlation analysis, we did not detect significant correlations between CP volume and total semen volume (r 5 0.16, P 5 0.59) or the total number of sperm cells (r 5 20.09, P 5 0.76) at T0–T4. Mean semen volume at T0 (4.4 6 0.8 ml, N 5 15) represented 4.6% of mean CP volume (95.14 6 38.77 mm3), and mean total semen volume collected from T0–T4 (13.55 6 14.84 ml) represented 14.2% of CP volume. DISCUSSION

Despite theoretically strong selection to produce large numbers of sperm cells, the number of sperm cells we obtained from male Tree

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Swallows decreased by nearly 80% after being sampled hourly for four hours (Table 1). Our frequency of sampling was twice the per-hour rate of within-pair copulation attempts observed by Venier and Robertson (1991), implying that greater than average copulation rates may not completely deplete Tree Swallow sperm reserves. However, the number of sperm cells that males are able to transfer to females during copulation may be limited. After initial samples were obtained at T0, up to one third of all samples were either devoid of sperm or lacked semen (Fig. 1). Regardless of semen collection technique, it is not unusual to fail to obtain semen every time collection is attempted (Gee and Temple 1978). Therefore, our results imply that some Tree Swallow copulations may not result in the transfer of sperm. Copulations that do not result in sperm transfer have been observed in several species (Penquite et al. 1930; Birkhead 1991; Birkhead et al. 1995; Westneat et al. 1998) and have usually been attributed to sperm depletion. Sperm concentrations of individual males were unaffected by repeated sampling. Similarly, repeated sampling regimes using cloacal massage did not affect sperm concentrations in domestic Turkey (Meleagris gallopavus) breeder males (Noirault and Brillard 1999). However, Noirault and Brillard did not collect semen as frequently as did we. In contrast, frequent collections of semen have resulted in reduced sperm concentration in other species (Gee and Temple 1978). Manually expressing semen from the seminal glomera may not provide an accurate estimate of natural ejaculate volume (Pellatt and Birkhead 1994). For example, in Muscovy Ducks (Cairina moschata) the volumes of ejaculates collected by cloacal massage are smaller than those collected by enticing males to ejaculate into a false cloaca (Kamar 1962; Gvaryahu et al. 1984; Samour et al. 1985). The semen volumes we obtained by cloacal massage from Tree Swallows were comparable to those obtained from similarly-sized male Bark Swallows (Riparia riparia) that were enticed to copulate with a model female fitted with a false cloaca (Nicholls et al. 2001). In contrast, Tree Swallow semen volumes we obtained by cloacal massage were smaller than semen volumes similarly obtained from smaller Splendid (M. splendens melanotus) and White-winged (M. leucopterus lu-

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conotes) fairy-wrens (Tuttle et al. 1996). FairyWrens experience very intense sperm competition and have extremely high rates of extra-pair paternity (Tuttle et al. 1996) that favor the production of large ejaculates and high sperm concentrations. Comparisons between the ejaculate volumes of Tree Swallows and other passerines are precluded by the lack of precise data on either natural ejaculate volumes or semen volumes collected by cloacal massage. What little data do exist (Gee and Temple 1978) suggest that most passerine ejaculates are # 10 mL. During each sample period, we collected semen samples until we were unable to express any more semen from the cloaca. Nevertheless, we were still able to obtain subsequent samples from some, but not all, males (Fig. 1). This result suggests that our sampling method may have depleted the sperm reserves found in the distal ends of the seminal glomera and that that area was subsequently refilled with sperm stored more proximally during a ‘‘refractory period.’’ A similar mechanism could also be responsible for the pattern of sperm depletion and replenishment observed in male Red-winged Blackbirds (Agelaius phoeniceus; Westneat et al. 1998). Our inability to obtain consecutive samples of equivalent sperm content from some individuals indicates that there may have been individual variation in the length of the ‘‘refractory period,’’ producing variation among males in their ability to transfer large numbers of sperm during a copulation that follows shortly after another. Temporary sperm depletion could negatively affect a male’s ability to prevail in sperm competition against a competitor with larger sperm reserves. The total number of sperm cells we collected from male Tree Swallows varied by orders of magnitude. Indeed, 40% of males either produced at least one sample lacking sperm cells or failed to produce a semen sample (Fig. 1). Variations in sperm counts among males and/ or among successive ejaculates from individual male passerines have been detected in several species (e.g., Birkhead et al. 1994; Møller 1994; Pellatt and Birkhead 1994; Tuttle et al. 1996; Kast et al. 1998; Westneat et al. 1998; Nicholls et al. 2001; Pizzari et al. 2003). In birds, infertility has been associated with depleted sperm supplies (Birkhead and Møller 1992; Kast et al. 1998). In our study, semen samples lacking sperm may have been due to sperm depletion

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from repeated sampling. Semen samples devoid of sperm may have also resulted from the release of fluids from the lymphatic folds and erectile tissues in birds during cloacal massage (Gee and Temple 1978); thus, cloacal massage may result in overestimating semen volume. However, this was unlikely in our study because there were no significant differences among the sperm concentrations in hourly samples from individual males. We think that collecting consecutive semen samples by cloacal massage does provide a useful lower estimate of the number of sperm stored in the seminal glomera. Directly testing this idea in Tree Swallows would require sampling males until their reserves appear to be depleted and then sacrificing them to count any sperm cells that might remain in the seminal glomera. Using this method, Birkhead et al. (1995) found that 84% of Zebra Finch (Taeniopygia guttata) sperm reserves are available for future copulations, and Australian Fairy-wren semen samples collected by manual expression contained 47% of the total number of stored sperm (Tuttle et al. 1996). The variation in semen volumes we obtained from male Tree Swallows may have resulted from variation in sampling effort. However, we attempted to control for this potential confounding factor because the same individual collected all samples and the same sampling techniques were used each time samples were collected. Therefore, we think that the differences in the total number of sperm cells we obtained from male swallows reflected individual differences among them in the size of their sperm reserves. We were unable to detect statistically significant relationships between the number of total sperm we obtained from males and their morphology, the date samples were collected, or the availability of reproductive opportunities the day a sample was collected. Our inability to detect significant relationships may be the result of low statistical power because of relatively small sample sizes. Regardless of its cause(s), individual variation in male responses to frequent semen sampling has important implications for sperm competition in Tree Swallows. Because success in sperm competition is influenced by the number of sperm in an ejaculate (Martin et al. 1974; Birkhead and Møller 1992), the differences among male Tree Swallows in the number of

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sperm cells that we collected from them suggests that some male swallows may be at a disadvantage relative to others in sperm competition. Our results may be especially relevant to the ability of a male to prevail in sperm competition during EPC because within-pair copulations are rare while their mates are incubating (Venier and Robertson 1991; Robertson et al. 1992). Additionally, the high frequency of extra-pair paternity in Tree Swallows could result from differences among males in sperm availability with low sperm-producing males being cuckolded more frequently than are high sperm-producing males. Sperm concentrations of individual males also varied during pre-laying and egg laying periods (Lombardo et al. 2002). We did not control for the copulation behavior of the males in our study. Therefore, the differences in their responses to frequent sampling could have been due to their copulation behavior prior to our capturing them. For example, the males from whom we were unable to obtain five semen samples may have had their semen reserves depleted by successful EPC before we sampled them. This possibility underscores our main point that copulation may affect a male Tree Swallow’s ability to successfully participate in subsequent inseminations. Kempenaers et al. (1999) found that extrapair male Tree Swallows had larger CP, and inferred that they had larger sperm reserves than did the males they cuckolded, probably because the sperm reserves of extra-pair males were not depleted by frequent copulations. This interpretation implies that the numerical advantage of the sperm from extra-pair males allowed them to out-compete the sperm of the males they cuckolded (Kempenaers et al. 1999). Interestingly, these extra-pair males were not more successful at avoiding being cuckolded at their own nests (Kempenaers et al. 1999). Changes in semen quality in response to repeated sampling may have important consequences on copulatory behavior in species in which both EPC and paternal care are common. From a mated male’s perspective, sperm depletion that results from his successful pursuit of EPC may increase his risk of being cuckolded if his social mate has also participated in EPC because he may not be able to inseminate his mate with enough sperm to out compete the sperm of his competitors. For example, in 1999 we observed a male copulating with his

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mate during the egg-laying period. We were unable to obtain a semen sample for him less than one hour later, suggesting that there is a ‘‘refractory period’’ after ejaculation during which sperm supplies are, at least temporarily, depleted (Lombardo et al. 2002). The potential for sperm depletion should favor the prudent allocation of ejaculates by males (Dewsbury 1982). Ade´lie Penquin (Pygoscelis adeliae) males strategically allocated ejaculations between their mates and extra-pair partners by withholding ejaculates from their mates (Hunter et al. 2000). Male Bank Swallows increased the sperm content of their ejaculates during copulations with model females fitted with false cloacae when there was an increased risk of sperm competition (Nicholls et al. 2001). Male domestic fowl (Gallus gallus) varied the sperm content of their ejaculates depending on their own social status, the risks of sperm competition, their familiarity with their copulation partner, and the relative size of their partner’s sexual ornaments (Pizzari et al. 2003). In contrast, there was no evidence of prudent allocation of ejaculates by male Red-winged Blackbirds during successive copulations with a model female fitted with a false cloaca (Westneat et al. 1998). However, the prudent allocation hypothesis was not directly tested. From a faithfully mated female’s perspective, being mated to a high-quality male who obtains many EPC may affect her fertility because her mate may not inseminate her with enough sperm to ensure her fertility. These circumstances would favor females that seek EPC. Female Red-winged Blackbirds may seek EPC as fertility insurance because females mated to males with many copulatory partners had more unhatched eggs in their nests (Gray 1997). Krokene et al. (1998) argued that the function of EPC in Great (Parus major) and Blue (P. caeruleus) tits could be fertility insurance if a small proportion of mated males were infertile or had low sperm counts. Determining if Tree Swallow males prudently allocate ejaculates and females pursue EPC as fertility insurance to compensate for the sperm depletion of their mates requires further investigation, as does determining the effects of individual variation in the size of sperm reserves on patterns of extra-pair paternity.

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ACKNOWLEDGMENTS

We thank C. R. Brown, B. Kempenaers, M. S. Webster, and anonymous reviewers for comments on previous versions of the manuscript. This study was supported by a 2000 Summer Undergraduate Research Program grant from the Science and Mathematics Division to MLG, MPL and PAT and the Department of Biology at Grand Valley State University. Czarnowski was supported by a New Jersey Agricultural Experiment Station Hatch Fund Grant to HWP at Rutgers University. The Institutional Animal Care and Use Committee at Grand Valley State University approved this study.

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