Disruption of ejaculates by male copulation in deer mice (Peromyscus maniculatus)

Physiology & Behavior, Vol. 41, pp. 53--58.Copyright©PergamonJournals Ltd., 1987. Printed in the U.S.A.

0031-9384/87 $3.00 + .00

Disruption of Ejaculates by Male Copulation in Deer Mice

(Peromyscus maniculatus) DONALD

A. DEWSBURY, 1 LAWRENCE

E. S H A P I R O A N D S T E P H E N

A. T A Y L O R

U n i v e r s i t y o f Florida R e c e i v e d 29 A p r i l 1987 DEWSBURY, D. A., L. E. SHAPIRO AND S. A. TAYLOR. Disruption of ejaculates by male copulation in deer mice (Peromyscus maniculatus). PHYSIOL BEHAV 41(I) 53-58, 1987.--Six experiments were conducted to analyze possible disruption of sperm transport occurring when one male deer mouse copulates soon after another male ejaculates. When a second male mated soon after the first, females had significantly fewer uterine sperm 1 hr later than when a delay of 7 or 15 min was imposed between males. However, it was impossible to detect any effect of such disruption on pregnancy initiation. Similarly, artificial stimulation soon after an ejaculation was ineffective in disrupting pregnancy initiation. No effect of a rapid resumption of copulation could be detected on litter composition in a sperm competition situation. Although it is clear, from these and other data, that the phenomenon of ejaculate disruption occurs in deer mice, technical difficulties may interfere with experimental analysis. Peromyscus maniculatus

Sperm transport

Pregnancy

IN the normal copulatory behavior of a single male-female pair of laboratory rats each ejaculation by the male is followed by a refractory period lasting 5 min or more during which no copulations occur [4]. Adler and Zoloth [1] conducted a series of studies of the effects of vaginal stimulation received by the female during this period of normal quiescence. Soon after a female received an ejaculate from one male they either allowed a second male to mate with her, either for five intromissions or for one ejaculation, or they provided artificial vaginal stimulation, with the barrel of a plastic syringe. Adler and Zoloth found that vaginal stimulation soon after receipt of an ejaculate interfered with the effectiveness of that ejaculate, relative to control animals, in three ways. First, fewer of the first male's sperm reached the uterus. Second, if the second male was not permitted to ejaculate, fewer females became pregnant. Third, if the second male was permitted to ejaculate, the first male sired fewer offspring than if a 15 min delay was imposed. Adler and Zoloth concluded that vaginal stimulation soon after an ejaculation disrupts sperm transport. These results were followed up by Matthews and Adler [22]. The disruption appears to be due, at least in part, to the dislodging of the copulatory plug deposited at ejaculation [2,17]. The phenomenon of ejaculate disruption has had appreciable impact on thought concerning the evolution of reproductive behavior (e.g., [20,21]). However, it has received little experimental analysis. The phenomenon has not been

Sperm competition

Copulation

documented experimentally in species other than laboratory rats and there has been no further analysis in rats. The present research was designed to provide an experimental analysis of ejaculate disruption in deer mice, Peromyscus rnaniculatus . It is known that female deer mice copulate with more than one male during a single estrous period in the field; multiple paternity of litters conceived in the field has been demonstrated [5]. Similar behavior has been observed in seminatural enclosures [9,11]. The occurrence o f ejaculate disruption in deer mice can be inferred from a correlational analysis of data collected by Dewsbury [12]. Single females copulated with two males. When all ejaculations were considered, the male ejaculating more sired more, but not significantly more, offspring than the male ejaculating fewer times. If all ejaculations after which the second male copulated within 1 rain or less were discarded from the analysis, however, the predictability of litter composition was improved and the difference between the two males was statistically significant. The phenomenon was cross-validated with two additional data sets [9,12]. This suggests that copulations by a male occurring soon after ejaculations by another male can effectively cancel effects of the first male's ejaculation. However, all of these data are correlational; the phenomenon requires experimental analysis. The phenomenon is of great importance for the evolution of reproductive behavior. Evolution is driven by natural

1Requests for reprints should be addressed to Donald A. Dewsbury, Department of Psychology, University of Florida, Gainesville, FL 32611.

53

54

DEWSBURY, SHAPIRO AND TAYLOR TABLE 1 DESIGN OF SIX EXPERIMENTS ON EFFECTS OF E J A C U L A T E D I S R b P T I O N

Experiment 1 2 3 4 5 6

Dependent Variable N sperm Pregnancy Pregnancy Pregnancy Sperm comp. Pregnancy

Mm Between Males C, 0, 7, 15 0, 10 0, 15 0, 15 0, 15 0, 15

2nd Male Stim. 5 Is Amf. 5 Is 5 Is I Ejac. Amf.

selection, with genetic fitness defined as "The contribution to the next generation of one genotype in a population relative to the contributions of other genotypes" ([27], p. 585). The phenomenon of sperm competition, the competition of ejaculates from two or more males mating with a single female [23], lies at the heart of differential reproduction and fitness. If a male can disrupt the reproductive processes of other males and facilitate his own, such behavior should be favored by natural selection. Damselflies, for example, have evolved specialized penile structures for the removal of sperm deposited by other males [26]. Similarly, the mutlipleintromission copulatory pattern and associated penile reflexes of laboratory rats may have evolved, at least in part, for the removal of copulatory plugs, and possibly associated sperm, deposited by other males [10,18]. In all, six experiments were conducted. Experiment 1 was designed to obtain data on the effect of disruptive stimulation on sperm counts. Experiments 2, 3, 4, and 6 were designed to study the effects of stimulation on the process of pregnancy initiation, with artificial vs. copulatory stimulation and cycling vs. postpartum estrus in a 2x2 array in the four experiments. Experiment 5 was designed to assess possible effects of disruption in the context of sperm competition. METHOD

Subjects Subjects were male and female deer mice, Peromyscua maniculatus bairdi, that were laboratory born and at least 90 days of age at the beginning of testing. They were from stock orignially trapped in the 1970's near Lansing, Michigan, but into which wild stock had been bred on several occasions. Mutant female deer mice of the blonde phenotype [24] were used as non-experimental mating partners in several studies. Mice were maintained in a windowless, airconditioned room on a reversed 16:8 light/dark photoperiod of white fluorescent light with light offset at 0930 hr and onset at 1730 hr. Animals were housed in clear polycarbonate cages, measuring 48x27x13 cm for males and 29x 19x 13 cm for females. Wood shavings were provided as bedding and water and Purina laboratory rodent chow were continuously available. All wild-type animals used were known to be homozygous for one of the two alleles (slow or fast) affecting the rate of migration of transferrin (Trf), as determined with horizontal starch-gel electrophoresis. These will be referred to as " S " and " F " males and females. The genotypes of each

N Females

N Test, per Condmon

48 12 21 21 23 34

12, 12. 11, 13 12, 12 12, 10 12, 12 12, 12 34. 34

Design between within between between between wahm

[~'~11 US

cychng cychng cychng ppartum ppartum ppartum

adult, and each pup in Experiment 5, were checked using methods described by Dewsbury [13]. All wild-type animals were of proven fertility. Some bore or sired litters in an earlier experiment. Others were paired for 15-20 days, with only those animals producing litters included in the study. Animals were sometimes used in more than one study.

Procedures In the basic paradigm used in each experiment, an experimental female was introduced into the home cage of a male and permitted to mate with him for one complete ejaculatory series. She was then removed, restrained for a predetermined time interval, and then either permitted to mate with a second male or given artificial vaginal stimulation. All tests were conducted during the dark phase of the diurnal cycle in dim red light. Six experiments were conducted; design features of individual experiments are summarized in Table 1. If the second-male stimulation was to be a complete ejaculatory series, the female was simply introduced into the second male's home cage and permitted to mate for the one series. In other experiments, the second males mated for just five intromissions, with no ejaculation, and females were removed immediately after the fifth intromission. If a male ejaculated after fewer than five intromissions the test was discarded. When artificial stimulation was called for, a "simulated series" was used with insertions of the blunt end of a Grumbacher No. 615 artist's paint brush (see [8]). For studies with females in cycling estrus, daily vaginal smears were taken from a bank of females using tap water and a thin wire loop. Females were mated on the first day with a smear lacking leukocytes, generally with predominantly nucleated epithelial cells. Because of the higher incidence of pregnancy [8] and relevance to behavior in nature [6], some studies were conducted with females in postpartum estrus. For such studies, females that had either been paired with a male to be tested for fertility or that had mated with a male in another experiment were checked each morning for the presence of a new litter. Tests were conducted on the afternoon of a day on which a litter was found. Where estrus was hormone-induced, females were injected intramuscularly with 0.06 mg estradiol benzoate 3 days before testing and 0.6 mg progesterone on the morning of testing. If the dependent variable was the incidence of pregnancy, each female's home cage was checked daily for the presence

55

E J A C U L A T E DISRUPTION IN DEER MICE TABLE 2 NUMBERS OF SPERM AND RESULTSOF MANN-WHITNEYU-TESTSFOR EXPERIMENT 1 Vs. Control

Condition

Vs. 7 Min

V s . 0 Min

Sperm × 106

U

z

p

U

z

p

U

z

p

12.20 6.86 14.62 15.27

20 38 44

-1.85 0.25 0.27

0.06 n.s. n.s.

31 40

-2.15 2.07

0.03 0.04

71

-0.03

n.s.

Control 0 min delay 7 min delay 15 min delay

40

o

x

3o

• -.. Oz

; .:

..:" .%

•

I0" I

o

500

-i

I00O

i

1500

I

2000

I

2500

I

3000

DELAY BETWEEN M A L E S ( s e c ) FIG. 1. Number of sperm recovered in the uterus of females in the 0-, 7-min, and 15-min delay groups as a function of the total delay between the first male's ejaculation and the second male's first intromission (imposed delay plus the second male's intromission latency).

of a litter beginning on Day 23 after mating. In Experiment 5, in which sperm competition was studied, the two males were homozygous for opposite Trf genotypes and each male mated with the experimental female for one ejaculation. When a litter was born, the genotype of each individual was determined using methods described by Dewsbury [13]. Where sperm counts constituted the dependent variable, the female was sacrificed 1 hr after receiving an ejaculate and counts of uterine sperm were made using methods described by Austin and Dewsbury [3] in their Experiment 1. The intervals imposed between males varied from 0-15 min (see Table 1). In the additional control condition (C) of Experiment 1, the female received no additional mating or stimulation after mating for one ejaculatory series. Between 12 and 48 females served in each experiment. In some studies with between-subjects designs a few animals served in more than one condition; the same animal never served twice in the same condition, however. A bank of 61 males, of the S or F genotype, were used as mating partners. Counterbalanced orders of test conditions were used in all studies with within-subjects designs. Deer mice are generally somewhat slow in initiating copulation [7]. In the present study, however, it was necessary that the second male mate as soon as possible after introduction of the female. To facilitate initiation of copulation, therefore, the males designated as " s e c o n d " males first mated for one complete ejaculatory series with a hormone-

induced, non-experimental, blonde female. A delay of at least 10 min was then allowed before introduction of the experimental female. The reuse of highly experienced males also facilitated the rapid initiation of copulation. Behavioral Measures Patterns of copulatory behavior were recorded on an Esterline-Angus operations recorder. There are three primary classes of events in the copulatory behavior of deer mice: mounts (without vaginal penetration), intromissions (with vaginal insertion and no sperm transfer), and ejaculations (with insertion and sperm transfer). These events are organized in "series," each of which begins with an intromission, ends with an ejaculation, and is separated from other series by a post-ejaculatory refractory period. The standard measures were taken: mount latency (ML), time (sec) from start of a test to the first mount or intromission; intromission latency (IL), time (sec) from start of a test to the first intromission; ejaculation latency (EL), time (sec) from the first intromission of a series until the ejaculation; intromission frequency (IF), the number of intromissions in a series; mount frequency (MF), the number of mounts in a series; mean inter-intromission interval (MIII), the mean interval (sec) separating the intromissions of a series; and post-ejaculatory interval (PEI), time (sec) from an ejaculation until the next intromission (see [7]).

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DEWSBURY, SHAPIRO AND TAYIX)R TABLE 3 I N C I D E N C E O F S U C C E S S F U L P R E G N A N C Y IN S T U D I E S OF E J A C U L A T E DISRUPTI()N

Percent Pregnant

[.liter size

Ge,,t. Ida} ',J

Delay Short Long

I)ela~ Short Long

Actual Delay Delay Experiment

Short

Long

2 3 4 5 6

62.2 24.6 38.3 33.7 20.9

681.9 922.1 915.9 920.6 888.6

Long

ChF

41 ~5/12~ 40 (@10) 83110/12) 81f13/16) 82128/34~

3.09 0.10 0.00 0.74 0.72

Short 50 (6/12) 33 (4/12) 83(10/12) 92(12/13) 79(27/34)

62 5.0 5.5 5.5 5.4

23.3 23.7 25.9 26.2 24.6

23.(I 23.0 24.8 25.4 24.6

rameters of copulatory behavior (ML, IL, EL, IF, MF, and Mill) by the ejaculating male. There were no significant correlations.

TABLE 4 R E S U L T S O F E X P E R I M E N T 5: E F F E C T S O F D E L A Y S ON

SPERM COMPETITION Mean After Delay

5.2 3.8 5.7 5.9 5.0

Mann-Whitney U-Tests

Measure

Short

Long

U

z

p

N litters N pups born N pups male 1 N pups male 2 Percent pups male 1 Pups: M1 vs. M2: Wilcoxon T z p

12 69 37 32 54.3

12 68 26 42 39.7

64 58 58 58.5

0.76 0.81 0.81 0.78

n.s. n.s. n.s. n.s.

35 0.31 n.s.

25.5 1.06 n.s.

I

Pregnancy Initiation Data on the incidence of pregnancy in Experiments 2-6 are presented in Table 3. There were no significant effects of the length of the postejaculatory quiescent period on the probability of successful pregnancy initiation. This was true for both tests with the second male mating for five intromissions (Experiments 3 and 4) and with artificial stimulation (Experiments 2 and 6). Pregnancy initiation was generally more reliable with females in postpartum estrus (Experiments 4-6) than in cycling estrus (Experiments 2-3) (see [8]). There were no significant effects of delay on either litter size or gestation period. Gestation periods were generally longer in postpartum estrus.

Sperm Competition RESULTS

Sperm Counts Data on sperm counts in Experiment 1 are presented in Table 2. The range of uterine sperm counts in the control, 7-min, and 15-min conditions was 12-16x 106. The mean value for tests with a second male mating after no delay was approximately half of these values, 6.86x106. A KruskalWallis analysis of variance done to compare the 0, 7, and 15 min delay groups revealed a significant difference, H(2)=6.00, p=0.048. When the control condition was included, the difference fell just short of significance, H(3)=6.34, p=0.094. As can be seen in Table 1, MannWhitney U-tests showed the 0-min condition to be significantly different from both the 7-min and 15-min conditions. Because deer mice require some time to initiate copulation, there was some delay after introduction of the second male until his first intromission occurred (means=275-434 sec for different conditions). The result of this effect plus the imposed delays of 7 and 15 min is a nearly continuous distribution of intervals between the first male's ejaculation and the first intromission by the second male. A scatter plot of these data is presented in Figure 1. It can be seen that sperm counts increase with increasing delays. The Pearson product-moment correlation between sperm counts and total delay was +0.42, t=2.72, p=0.01. Correlational analyses were used to search for a relationship between the number of sperm recovered and the pa-

Data from Experiment 5 on sperm competition are presented in Table 4. With a short interval between males the first males sired 54% of the pups; the first males sired 40% with the long delay. In neither condition was there a significant first- or second-male advantage (Table 4). Further, there was no significant difference in litter composition between the groups.

Copulatoo' Behavior Data on the parameters of copulatory behavior were examined for all six experiments. They were generally within the normal range for this population (e.g., [70, with no obvious experimental effects. DISCUSSION

It is clear that ejaculate disruption occurs in deer mice. If a female receives five intromissions without an ejaculation as soon as possible after she receives an ejaculate, the number of sperm in her uterus is significantly reduced in comparison to the number that would be present if a delay of 7 or 15 rain was imposed. Further, there is good correlational evidence that these disruptions are functional. In the study of Dewsbury I12] the predictability of litter composition from the patterning of ad lib ejaculatory behavior of two males mating with a single female was better if those ejaculates not followed by a quiescent period of at least 1 rain were eliminated from the analysis than if they were included. Deer mice are the only species in which ejaculate disruption has been demon-

E J A C U L A T E D I S R U P T I O N IN D E E R MICE

57

strated with two males mating in an ad lib situation, which may mimic behavior in the field. Deer mice are the first species other than laboratory rats, the species in which the phenomenon was first demonstrated, that has been shown to display the disruptive effect of copulation soon after an ejaculation. Attempts to demonstrate functional consequences of ejaculate disruption experimentally led to disappointing results. Although five intromissions soon after an ejaculation were sufficient to decrease sperm counts, there was no decrement in pregnancy initiation. This was true with females in both cycling estrus, where pregnancy rates after one ejaculation are relatively low (Experiment 3), and postpartum estrus, where pregnancy rates are higher (Experiment 4). Similary, although artifical stimulation, as provided here, can be effective in pregnancy initiation [8], it was without a disruptive effect on pregnancy in both cycling estrus (Experiment 2) and postpartum estrus (Experiment 6). When the second male was permitted to ejaculate, thus setting up a sperm competition situation, the delay between males was of no significant consequence for litter composition. Thus, whereas in rats there appear to be functional consequences of ejaculate disruption for sperm counts, pregnancy rates, and litter composition [1,22], only the first of these effects could be demonstrated experimentally in deer mice. It is likely that the reasons for these failures are technical; it is difficult to execute the appropriate experiments in deer mice for several reasons. First, the period of susceptibility appears quite short. The maximal effect in the correlational data of Dewsbury [12] was obtained at 1 rain. In the present data, sperm counts had already returned to baseline levels if delays of just 7 min were permitted before introduction of the second male. Because of their small size and considerable agility, it is difficult to move a female deer mouse from one cage to another with the speed with which one can handle a laboratory rat. Further, the latency for deer mice to begin copulating is generally much longer than that in laboratory rats [7]. For this reason, the experiments were designed so that the second male completed one ejaculatory series before mating with the experimental female. Although this procedure appeared to decrease the total intervals between males, the mean actual times from the first male's ejaculation to the second male's first intromission in tests with short intervals

were still actually 142.7, 202.1,178.1, and 140.9 s for Experiments 1, 3, 4, and 5 respectively. This may be too long, but it would be difficult to execute an experiment with shorter delays. Compounding these difficulties is the relatively low probability of pregnancy for females in cycling estrus after one ejaculatory series in this species [8]. This creates a floor effect, rendering it difficult to detect decrements in pregnancy initiation. Thus, the most likely interpretation of these data is that ejaculate disruption in deer mice is probably very similar to that in laboratory rats, but that technical difficulties interfere with experimental analysis. These difficulties are not a factor when males are mating ad lib and often copulate one right after the other [12]. Copulatory plugs were frequently disolodged by artificial stimulation soon after ejaculation when females were in postpartum estrus. These plugs appeared identical to those found in a previous study [19]. Plug formation must be quite rapid. Plugs were removed in most of the tests with short delays (mean=20.9 sec) in Experiment 6. Yet, there was no effect on pregnancy and thus plugs appear to play no role in sperm competition under these conditions [14]. The lack of an effect of I F on sperm counts in the present data contrasts with data on laboratory rats [25]. In the present study all tests were run ad lib with a single pair mating at a time. This may partially explain the different result. The similarity of many fine points of the data collected in this study to previously reported results suggests than no major procedural difficulty was encountered. Quantitative parameters of copulatory behavior were in the normal range (see [7]). Sperm counts for the first ejaculate were close to values previously reported [16]. Percentages of females pregnant resembled earlier values and females were more likely to become pregnant when in postpartum than in cycling estrns (see [8]). The lack of an order effect in sperm competition is consistent with previous results when both males attain equal numbers of ejaculates under these conditions [13,15]. ACKNOWLEDGEMENTS This research was supported by Grant BNS-8520318 from the National Science Foundation. I thank Mrs. Susan Hoffmann for technical assistance with the electrophoretic procedures and Dianalee Deter and Joyce O. Sanders for doing vaginal smears.

REFERENCES

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