Physiology & Behavior, Vol. 47, pp. 483--488.©PergamonPress plc, 1990. Printedin the U.S.A.
0031-9384/90$3.00 + .00
The Effect of Time of Day on Sperm Competition and Male Reproductive Success in Laboratory Rats M. ~ P I N K A Research Institute o f Animal Production, CS-I04 O0 Prague lO-Uh[fn~ves, Czechoslovakia Received 28 March 1989
~PINKA, M. The effect of time of day on sperm competition and male reproductive success in laboratory rats. PHYSIOL BEHAV 47(3) 483-488, 1990.--The study was designed to test the hypothesis that siring success of male rats depends on when during the receptivity period the matings occur. In Experiment 1, females were mated with pairs of males differing in coat colour genetic marker. The males copulated with the female successively, either about 1 hour apart at dusk (schedule AA) or about 1 hour apart around midnight (BB) or one at dusk and the other around midnight (AB). The males mating first had lesser siting success under the schedule AB than under the other two schedules. In Experiment 2, females were mated under the same regimen as in Experiment 1. One male of each competing pair had its spermatozoa labeled by 3H-thymidine. The proportions of spermatozoa of the labeled males were identified in samples from uterine horns and oviducts. Balanced conaibutions of both males were recorded in uterus in all groups. No definitive conclusion concerning oviductal sperm composition could be reached. In both experiments, night-mating males copulated more quickly. It is suggested that copulation at about midnight is advantageous in intermale reproductive competition and that the function of the diurnal variation in the speed of male sexual behaviour might be clarified on this ground. Male sexual behavior
Paternity
Sperm competition
Sperm labeling
GIVEN the opportunity, females of some rodents species copulate with more than one male during a single mating period, and more males may father a given litter (5). A considerable amount of research has been focused on the relationship between copulatory behaviours during multiple-male mating and reproductive success of the males involved (1, 5, 7, 8, 14). Three parameters of multiple-male mating relating to the timing of copulations have been implicated to influence the compositions of litters. First, paternity may be influenced by the order of mating. A "first male" advantage has been observed in house mice and guinea pigs (5). In laboratory rats, Lanier et al. (14) reported an absence of order effects. A second male advantage was found in prairie voles, Microtus ochrogaster, by Dewsbury and Baumgardner (6). Second, the role of time delay between ejaculations of either male has been studied. The outcome of competition was not changed by imposing a 2-hr delay between matings of two deer mice males (6). Huck et al. (12) increased the time interval between matings of male hamsters from 30 min to 8 hr and demonstrated increasing siring success of the first male. Last but not least, the competition may be influenced by when matings occur. Huck et al. (13) found different composition of litters when two hamster males mated with the same delay but at different times relative to ovulation. The last study also demonstrated that there is an important interaction of the three aspects in hamsters; e.g., second male mating advantage was recorded in matings at the beginning of the receptivity period, whereas mating later resulted in a first male advantage. The roles of time delay and time of mating have not been
Rats
investigated in rats. Two points suggest that it may be important. First, cycling laboratory rats ovulate spontaneously between 02:00 and 05:00 a.m. (28) and are receptive over a period of about 14 hours starting 9-12 hours before ovulation (10,20). It follows that the residence time of spermatozoa within a female reproductive tract may differ considerably between males due to the long period of receptivity in the female rat. The variable residence time may, in turn, affect sperm motility (9) and/or fertilizing ability (3). Second, male rats copulate progressively faster as the afternoon and the night advances (4, 11, 26) and female rats add in the course of the receptive period new components to their proceptive behaviour that are more stimulating for males (16,17). It is felt that these gradients in behaviour have some biological function related to individual reproductive success. The present experiments were designed to examine the importance of the time factors for reproductive success (Experiment 1) and to suggest a possible mechanism of such an effect (Experiment 2). For this purpose, each experimental female was mated to two males under one of three time regimens, which may to some extent parallel those occurring in nature: a female rat may accomplish all its mating either at the beginning of the receptive period in the evening, or much later just before ovulation at about midnight, or it may prolong its copulation and so receive part of the ejaculations in the evening, and part in the night. EXPERIMENT 1 The main purpose of Experiment 1 was to evaluate the effect of time of mating relative to the expected time of ovulation on the
483
484
reproductive success of males. The basic research strategy was that used by numerous previous investigators (1, 7, 14, 23). A female of known recessive genotype is allowed to copulate with males of two strains, one of them homozygous for the dominant and the other for the recessive allele. Males of two strains may, however, differ in the relative ability to gain representation in litters sired, when their sperm are deposited in the same female reproductive tract under balanced conditions. This differential fertilizing capacity (DFC) represents, in fact, a common finding in studies of this type (5). In order to avoid this complication, congenic males of an inbred strain were used. Method
Female subjects were locally bred Wistar albino rats. Females displaying 4-day cycles were employed. They were 62-152 days old at the beginning of the experiment and, except for 3 primiparas, they were all virgin. Experimental males were all F 3progenies of crossings between albino Lewis females from the Section of Animal Experimental Models, Czechoslovak Academy of Sciences, Prague, and congenic hooded LEW.C4A/Cub males from Faculty of Medicine, Charles University, Prague. Nine albino and 6 hooded homozygote males were used, the genotypes of the latter having been previously checked by back-crossing with albino females. They were all sexually experienced, 120-200 days of age when used. All animals had been living at least 4 weeks prior to testing in a room maintained on a 14:10 LD cycle. For convenience in comparison all times cited in the text refer to the rats' photoperiod, where 12:00 is the midpoint of the light period. Animals were paired within the room in a rectangular 1 square metre arena. In the subjective "morning," females with nucleated cells in the vaginal smears were selected. Prior to pairing, these females were left about 10 minutes in the arena to prescent it. The mating started with placing a male into the empty arena. Five minutes later, one of the selected females was introduced and left with the male until he ejaculated twice. The female was then kept in a separate cage and later was allowed to mate in the same manner with the second male homozygous for the other allele. Thus, each female received two ejaculations from an albino and two from a pigmented male. The times of the copulatory events were recorded with pen and paper. Females were assigned to one of three groups (12 subjects per group) which differed with respect to times at which the copulations were accomplished. Females in Group AA1 were mated with the first male just before lights-off (median of the first intromission: 17:30). Within an hour they were remated with the second male (median 1st intromission 18:42); the interval between the second ejaculation of the first male and the first intromission of the second male (further referred to as "delay between males") varied between 16-48 min, median 19 min. Females in the Group AB were assigned to coitus with the fn-st male at dusk (median 17:53), and with the second one around midnight (median 23:27, delay between males 281--407 min). The BB females were paired with the first male around midnight (median 23:27) and with the second one about one hour later (median 00:19, delay between males 26-58 min). Half of the females in each group were mated first to an albino male and then to a hooded male, the other half was mated in the reverse order. The time interval between two uses of the same male was at least 48 hr. Females were killed with an overdose of aether 15 days after insemination and uterine horns were inspected for number and eye colouration of viable fetuses. In the cases when less than 6 viable fetuses were found, the female was disregarded, and the respective group was completed by inseminating another female.
~PINKA
The following measures of male copulatory behaviour were computed: ejaculation latency (EL--time in minutes from the first intromission of a series until ejaculation); intromission frequency (IF--number of intromissions achieved prior to an ejaculation); interintromission interval (III--mean interval separating the intromissions within a series); postejaculatory interval (PEI--the time interval from an ejaculation to the first intromission of the next series). Variables referring to the first series of a male are denoted with number 1, those of the second series with number 2. Behavioural data were first logarithmized and then analyzed by nonrepeated ANOVA with a two factor design, time (dusk vs. around midnight) by order of copulation (1 st and 2nd vs. 3rd and 4th series from the standpoint of the female). Nonparametric statistical tests (24) were used for results on paternity. Results
All mated females experienced pregnancy. Three females of Group AA1 (i.e., those receiving all 4 ejaculations before dusk) had the majority of fetuses resorbing on the 15th postcoital day, with only 3, 4, or 5 viable fetuses remaining, respectively. Except for comparison of litter sizes among groups, these females were disregarded. The remaining 36 females had 9-17 nonresorbing fetuses on inspection. Results concerning litter size and siring success are given in Table 1. The groups did not differ in the median proportion of albino fetuses as indicated by Kruskal-Wallis ANOVA (K-W ANOVA, H = 1.201, N.S.). Percentages of albino offspring were pooled for all 36 females. Two hundred thirty-seven of the total 433 fetuses were albino (54.7%), which is not different from 50% (Wilcoxon T = 256.5, n = 36, N.S.). K-W ANOVA revealed a significant effect of the groups on the reproductive success of the 1st male (H = 6.867, n = 36, p
0031-9384/90$3.00 + .00
The Effect of Time of Day on Sperm Competition and Male Reproductive Success in Laboratory Rats M. ~ P I N K A Research Institute o f Animal Production, CS-I04 O0 Prague lO-Uh[fn~ves, Czechoslovakia Received 28 March 1989
~PINKA, M. The effect of time of day on sperm competition and male reproductive success in laboratory rats. PHYSIOL BEHAV 47(3) 483-488, 1990.--The study was designed to test the hypothesis that siring success of male rats depends on when during the receptivity period the matings occur. In Experiment 1, females were mated with pairs of males differing in coat colour genetic marker. The males copulated with the female successively, either about 1 hour apart at dusk (schedule AA) or about 1 hour apart around midnight (BB) or one at dusk and the other around midnight (AB). The males mating first had lesser siting success under the schedule AB than under the other two schedules. In Experiment 2, females were mated under the same regimen as in Experiment 1. One male of each competing pair had its spermatozoa labeled by 3H-thymidine. The proportions of spermatozoa of the labeled males were identified in samples from uterine horns and oviducts. Balanced conaibutions of both males were recorded in uterus in all groups. No definitive conclusion concerning oviductal sperm composition could be reached. In both experiments, night-mating males copulated more quickly. It is suggested that copulation at about midnight is advantageous in intermale reproductive competition and that the function of the diurnal variation in the speed of male sexual behaviour might be clarified on this ground. Male sexual behavior
Paternity
Sperm competition
Sperm labeling
GIVEN the opportunity, females of some rodents species copulate with more than one male during a single mating period, and more males may father a given litter (5). A considerable amount of research has been focused on the relationship between copulatory behaviours during multiple-male mating and reproductive success of the males involved (1, 5, 7, 8, 14). Three parameters of multiple-male mating relating to the timing of copulations have been implicated to influence the compositions of litters. First, paternity may be influenced by the order of mating. A "first male" advantage has been observed in house mice and guinea pigs (5). In laboratory rats, Lanier et al. (14) reported an absence of order effects. A second male advantage was found in prairie voles, Microtus ochrogaster, by Dewsbury and Baumgardner (6). Second, the role of time delay between ejaculations of either male has been studied. The outcome of competition was not changed by imposing a 2-hr delay between matings of two deer mice males (6). Huck et al. (12) increased the time interval between matings of male hamsters from 30 min to 8 hr and demonstrated increasing siring success of the first male. Last but not least, the competition may be influenced by when matings occur. Huck et al. (13) found different composition of litters when two hamster males mated with the same delay but at different times relative to ovulation. The last study also demonstrated that there is an important interaction of the three aspects in hamsters; e.g., second male mating advantage was recorded in matings at the beginning of the receptivity period, whereas mating later resulted in a first male advantage. The roles of time delay and time of mating have not been
Rats
investigated in rats. Two points suggest that it may be important. First, cycling laboratory rats ovulate spontaneously between 02:00 and 05:00 a.m. (28) and are receptive over a period of about 14 hours starting 9-12 hours before ovulation (10,20). It follows that the residence time of spermatozoa within a female reproductive tract may differ considerably between males due to the long period of receptivity in the female rat. The variable residence time may, in turn, affect sperm motility (9) and/or fertilizing ability (3). Second, male rats copulate progressively faster as the afternoon and the night advances (4, 11, 26) and female rats add in the course of the receptive period new components to their proceptive behaviour that are more stimulating for males (16,17). It is felt that these gradients in behaviour have some biological function related to individual reproductive success. The present experiments were designed to examine the importance of the time factors for reproductive success (Experiment 1) and to suggest a possible mechanism of such an effect (Experiment 2). For this purpose, each experimental female was mated to two males under one of three time regimens, which may to some extent parallel those occurring in nature: a female rat may accomplish all its mating either at the beginning of the receptive period in the evening, or much later just before ovulation at about midnight, or it may prolong its copulation and so receive part of the ejaculations in the evening, and part in the night. EXPERIMENT 1 The main purpose of Experiment 1 was to evaluate the effect of time of mating relative to the expected time of ovulation on the
483
484
reproductive success of males. The basic research strategy was that used by numerous previous investigators (1, 7, 14, 23). A female of known recessive genotype is allowed to copulate with males of two strains, one of them homozygous for the dominant and the other for the recessive allele. Males of two strains may, however, differ in the relative ability to gain representation in litters sired, when their sperm are deposited in the same female reproductive tract under balanced conditions. This differential fertilizing capacity (DFC) represents, in fact, a common finding in studies of this type (5). In order to avoid this complication, congenic males of an inbred strain were used. Method
Female subjects were locally bred Wistar albino rats. Females displaying 4-day cycles were employed. They were 62-152 days old at the beginning of the experiment and, except for 3 primiparas, they were all virgin. Experimental males were all F 3progenies of crossings between albino Lewis females from the Section of Animal Experimental Models, Czechoslovak Academy of Sciences, Prague, and congenic hooded LEW.C4A/Cub males from Faculty of Medicine, Charles University, Prague. Nine albino and 6 hooded homozygote males were used, the genotypes of the latter having been previously checked by back-crossing with albino females. They were all sexually experienced, 120-200 days of age when used. All animals had been living at least 4 weeks prior to testing in a room maintained on a 14:10 LD cycle. For convenience in comparison all times cited in the text refer to the rats' photoperiod, where 12:00 is the midpoint of the light period. Animals were paired within the room in a rectangular 1 square metre arena. In the subjective "morning," females with nucleated cells in the vaginal smears were selected. Prior to pairing, these females were left about 10 minutes in the arena to prescent it. The mating started with placing a male into the empty arena. Five minutes later, one of the selected females was introduced and left with the male until he ejaculated twice. The female was then kept in a separate cage and later was allowed to mate in the same manner with the second male homozygous for the other allele. Thus, each female received two ejaculations from an albino and two from a pigmented male. The times of the copulatory events were recorded with pen and paper. Females were assigned to one of three groups (12 subjects per group) which differed with respect to times at which the copulations were accomplished. Females in Group AA1 were mated with the first male just before lights-off (median of the first intromission: 17:30). Within an hour they were remated with the second male (median 1st intromission 18:42); the interval between the second ejaculation of the first male and the first intromission of the second male (further referred to as "delay between males") varied between 16-48 min, median 19 min. Females in the Group AB were assigned to coitus with the fn-st male at dusk (median 17:53), and with the second one around midnight (median 23:27, delay between males 281--407 min). The BB females were paired with the first male around midnight (median 23:27) and with the second one about one hour later (median 00:19, delay between males 26-58 min). Half of the females in each group were mated first to an albino male and then to a hooded male, the other half was mated in the reverse order. The time interval between two uses of the same male was at least 48 hr. Females were killed with an overdose of aether 15 days after insemination and uterine horns were inspected for number and eye colouration of viable fetuses. In the cases when less than 6 viable fetuses were found, the female was disregarded, and the respective group was completed by inseminating another female.
~PINKA
The following measures of male copulatory behaviour were computed: ejaculation latency (EL--time in minutes from the first intromission of a series until ejaculation); intromission frequency (IF--number of intromissions achieved prior to an ejaculation); interintromission interval (III--mean interval separating the intromissions within a series); postejaculatory interval (PEI--the time interval from an ejaculation to the first intromission of the next series). Variables referring to the first series of a male are denoted with number 1, those of the second series with number 2. Behavioural data were first logarithmized and then analyzed by nonrepeated ANOVA with a two factor design, time (dusk vs. around midnight) by order of copulation (1 st and 2nd vs. 3rd and 4th series from the standpoint of the female). Nonparametric statistical tests (24) were used for results on paternity. Results
All mated females experienced pregnancy. Three females of Group AA1 (i.e., those receiving all 4 ejaculations before dusk) had the majority of fetuses resorbing on the 15th postcoital day, with only 3, 4, or 5 viable fetuses remaining, respectively. Except for comparison of litter sizes among groups, these females were disregarded. The remaining 36 females had 9-17 nonresorbing fetuses on inspection. Results concerning litter size and siring success are given in Table 1. The groups did not differ in the median proportion of albino fetuses as indicated by Kruskal-Wallis ANOVA (K-W ANOVA, H = 1.201, N.S.). Percentages of albino offspring were pooled for all 36 females. Two hundred thirty-seven of the total 433 fetuses were albino (54.7%), which is not different from 50% (Wilcoxon T = 256.5, n = 36, N.S.). K-W ANOVA revealed a significant effect of the groups on the reproductive success of the 1st male (H = 6.867, n = 36, p
