Applied Animal Behaviour Science, 13 (1984/85) 371--381 371 Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands MATING BEHAVIOUR AND FERTILITY IN DOMESTIC CHICKENS. I. INBREEDING KIMBERLY M. CHENG 1, JEFFREY T. BURNS 2 and ROBERT N. SHOFFNER 3 1Avian Genetics Laboratory, Department of Poultry Science, University of British Columbia, Vancouver, B.C. V6T 2A2 (Canada) 2Department of Ecology and Behavioral Biology, University of Minnesota, Minneapolis, MN 55455 (U.S.A.) 3Department of Animal Science, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Accepted for publication 8 June 1984) ABSTRACT Cheng, K.M., Burns, J.T. and Shoffner, R.N., 1985. Mating behaviour and fertility in domestic chickens. I. Inbreeding. Appl. Anita. Behav. Sci., 1 3 : 3 7 1 - - 3 8 1 . Mating behaviour of males and females of the Inbred line 420 Single Comb White Leghorn chickens was compared with that of the random-bred Minnesota Marker line in a factorial experiment with two replications. Inbred males showed less frequency of approaching females, waltzing, treading and tall-bending, but did not differ from randombred males in frequency of attempted and successful mountings. Inbred females showed fewer crouchings and completed fewer matings than random-bred females. Fertility was associated with the interaction of male and female behaviour. INTRODUCTION Although it is well known that close inbreeding is nearly always accompanied by reduced fitness components (Johanson and Rendel, 1968), for a given inbred line, it is not predictable which aspect of performance will be most affected (Shoffner, 1948). The Minnesota 420 (I-420) is an inbred line of Single Comb White Leghorn chickens. The line was started in 1937 through full- and half-sib matings, with selection for egg production, egg and b o d y size (Shoffner et al., 1953). After the initial generations, the line has maintained a b o u t the same level of hatchability, egg production and b o d y size to the present time, although the coefficient of inbreeding is now above 90%. Since 1977, fertility through natural matings has declined to a point that natural matings must be supplemented by artificial insemination to propagate the line. An examination of semen from the 1-420 males showed that while volume per ejaculation is smaller, other semen characteristics such as sperm count, motility and percentage abnormalities are similar to those of random-bred populations (R.N. Shoffner, unpublished 0168-1591/85/$03.30 © 1985 Elsevier Science Publishers B.V. 372 data, 1979). On the other hand, casual observations showed that the mating frequency in this inbred line seemed to be very low. Comparisons of the 1-420 with a random-bred line afford the o p p o r t u n i t y to study mating behaviour of chickens with low libido, and to gain insight as to how inheritance affects mating behaviour. MATERIALS AND METHODS The populations studied were 1-420 line and the random-bred Minnesota Marker (MM) line (Shoffner, 1972). The MM line was established'by crossing eight different breeds of chickens and has been maintained as a random-bred closed population for the last 10 generations by the time of the experiment. This line of diverse origin provides a satisfactory control for the highly inbred 1-420. Birds used in the experiment were approximately 1 year old and had previous breeding experience. Although both lines were not raised as an intermingled flock, they were housed in adjacent pens or cages so that they had visual social experiences with each other. In each replication, 6 males and 16 females of each line were used. The females were separated from the males 2 weeks prior to the start of the experiment and were only put back together in the various treatment groups 2 days before the start of the observation period. Only laying females were used. The 8 treatment groups were: (1) one 1-420 male with four 1-420 females; (2) two I 4 2 0 males with four 1-420 females; (3) one MM male with four 1-420 females; (4) two MM males with four 1-420 females: (5) one 1-420 male with four MM females; (6) two 1-420 males with four MM females; (7) one MM male with four MM females; (8) two MM males with four MM females. Each group was placed in a 2.5 m × 2.5 m indoor pen with wood shavings litter. The sides of each pen consisted of a visual barrier of asphalt roofing material 61 cm high. The birds were exposed to a 14 : 10 h light--dark schedule (lights on 08.30 to 22.30 h) with water and feed provided ad libitum. Observations started 2 days after the birds were placed in the pens. During the next 14 days, five 30-min observations were made on each group during each of the following four time-periods: (1) Morning I: 09.00--10.00 h; (2) Morning II: 10.00--11.00 h; (3) Afternoon I: 19.00--20.00 h; (4) Afternoon II: 20.00--21.00 h; A total of 20 observations were made on each group, totalling 10 h. During the observation periods, all male--male and male--female interactions were recorded; in particular, the frequencies of the components of mating behaviour listed below. 373 (1) Approaching: male approaching female with head oriented towards her. (2) Pre-copulatory waltzing: male waltzing before, or without, mounting attempt. (3) Crouching: female crouching (receptive posture) before male attempts to mount. (4) Mounting attempt: male grasping female by the back of the head and attempting to m o u n t (at least one f o o t on female's back). (5) Successful mounting attempt: male has both feet on female's back. (6) Treading: male treads on female's back. (7) Tail-bending: male bending his tail around the female's tail. Tailbending was considered as an indicator of completed mating. In addition, eggs laid by the females during the experimental period were collected, identified by pen number, artificially incubated and hatched to determine fertility and hatchability. At the end of the 2-week period, a new set of birds was put in the pens and the experiment was repeated after the new birds were given 2 days in the pens for habituation. The cumulated frequencies (over 5 observations) of the 7 components of mating behaviour measured were analyzed separately by analysis of variance with the split-plot model (Snedecor and Cochran, 1980). Y~klm = P + R i + Mj + F k + e l + (MF)ik + (MC)]I + (FC)kl + (MFC)jkl + E1Ukl + Tm + (MT)jm + (FT)krn + (CT)lm + (MFT)jkm + ( F e T ) k l m + E2~klm and i = 1, 2 ; j = 1, 2; k = 1, 2;1 = 1, 2; m = 1, 2, 3, 4; where YUklm = one of the 7 dependent variables. Yi17~lm is the cumulated frequency of the behaviour in the ith replication, involving male(s) of the j t h line and females of the kth line in t h e / t h type of mating group during the ruth period of the day, p = the theoretical population mean, R i = effect of the ith replication, M i = effect of whether the male involved was from the 1-420 or the MM line, F k = effect of whether the female involved was from the 1-420 or MM line, Cl = effect of whether it was a 1-male or 2-male mating group, Tm = effect of observations made during the mth time period, (MF)jk, (MC)jT, (FC)kl, (MT)jm, (FT)km, (CT)Im = 2-factor interactions, ( M F T ) j k m , (FCT)klm, (MFC)jkl = 3-factor interactions, E l ~ k l = error term for main plot comparisons, and E2~iklm = sub-plot error term. The analyses were c o n d u c t e d with the aid of a computer program. " I V A N " (Weisberg and Koehler, 1979), at the University of Minnesota Computer Center. Square-root transformation was applied to "Crouching", "Treading" and "Tail-bending", the 3 variables with low frequency of occurrence, before the analyses. •: analysis was also used to test differences in mating efficiencies (e.g. tail-bending/successful mounting attempts) and proportions of behaviour patterns (e.g. waltz/approach). 374 RESULTS Only results relevant to line comparisons in mating behaviour are reported and discussed here. Competition between males and differences between the two types of mating groups will be discussed in another paper. Male behaviour 1-420 males approached and waltzed to females significantly less frequently than MM males (Table I). The difference in frequency of waltzes was still significant after the frequency of approaching was standardized, as the proportion of approaches with waltzes was significantly smaller (×2 = 4.74, d.f. = 1, P < 0.05) for 1-420 males (0.10) compared to MM males (0.20). Although 1-420 males approached and waltzed to females less frequently than MM males, they elicited the same number of crouches from the females as MM males. There were also no significant differences between the two types of males in either the frequency of mounting attempts or successful mountings. 1-420 males, however, treaded less frequently than MM males. Even after successfully mounting a female, 1-420 males often stood on the female's back briefly, and dismounted without completing the mating. As a consequence, the proportion of successful mounting attempts ending in tail-bending was significantly lower (X2 = 4.36, P < 0.05) for 1-420 males (0.44) than MM males (0.60). TABLE I Differences in f r e q u e n c i e s ~ o f m a t i n g behaviour b e t w e e n MM a n d 1 - 4 2 0 m a l e s Male genotype MM 1-420 Male m a t i n g b e h a v i o t t r Approach female Courtship waltz Crouch elicited Mounting attempts Successful mounting Treading Tailbending 10.89"* 7.37 2.21" 0.71 0.54 0.52 5.02 3.85 1.38 0.99 1.06" 0.67 0.83* 0.44 Frequency per male per 5 observations ( 2 . 5 h ) . *P < 0 . 0 5 ; **P "~ 0 . 0 1 . Female behaviour and effect o f female genotype on male behaviour Both MM and 1-420 females were approached and courted at the same frequencies by males (Table II), but 1-420 females were significantly less willing than MM females to crouch for males. The number of crouches per male approach was 0.03 for 1-420 females and 0.08 for MM females, with the difference being highly significant (×2 = 11.8, d.f. = 1, P ~ 0.005). As a result, there were significantly fewer successful mounting attempts on 1-420 than on MM females. Likewise, there was significantly less treading and 375 T A B L E II Differences in f r e q u e n c i e s ~ o f mating b e h a v i o u r b e t w e e n MM and 1-420 females Female genotype MM 1-420 Mating behaviour Approached by male Courted by male Female crouch Mounting attempts Successful mounting Treaded by male Tailbending 3.50 3.34 0.67 0.42 0.30** 0.10 1.67 1.66 0.59** 0.29 0.42** 0.22 0.34** 0.13 F r e q u e n c y per female p e r 5 o b s e r v a t i o n s ( 2 . 5 h ) . **P < 0.01. tail-bending by males on 1-420 than on MM females. The proportion of successful mountings ending in tail-bending for 1-420 females (0.46) and MM females (0.58) was not significantly different. Temporal patterns Differences in frequency of mating behaviour during different times of the day showed that males waltzed to females most often early in the morning, while mating activities peaked in the late afternoon when females were more willing to crouch (Table III). There appeared to be a lull in the mating activities during the latter portion of the morning. In terac tions There was a significant (P < 0.05) interaction between the genotype of the males and the time of day in the frequency of waltzes. Orthogonal comparisons showed that MM males waltzed significantly more often in the morning (both morning periods combined; 3.08) than in the afternoon (both afternoon periods combined; 1.33). 1-420 males did not show significant difference in frequency of waltzes in the same periods (0.88 vs. 0.54). There was, however, a significant (P < 0.05) difference in frequency of waltzes between MM and 1-420 males in the morning but not in the afternoon. The interaction of female genotype by time of day in the frequency of successful mounting attempts was also significant (P < 0.05). This interaction was because there were significantly more successful mounting attempts on MM females in the afternoon (0.88) than in the morning (0.44), while the frequency for 1-420 females remained low and was not different in the morning (0.26) and afternoon (0.44). There was a significant difference in the frequency of successful mounting attempts on the two types of females in the afternoon, b u t the difference was not significant in the morning. There was a significant (P < 0.05) 3-way interaction involving genotype of males, genotype of females and time of day in the frequency of waltzes. T A B L E III Differences in frequencies I o f m a t i n g b e h a v i o u r d u r i n g d i f f e r e n t t i m e s o f t h e d a y ( 0 8 . 3 0 - - 2 2 . 3 0 h ) Time Morning, 0 9 . 0 1 - - 1 0 . 0 0 Morning, 1 0 . 0 1 - - 1 1 . 0 0 Afternoon, 19.01--20.00 Afternoon, 20.01--21.00 Mating behaviour Approach** Waltz** Crouch** Mounting attempts** Successful mounting** Treading** Tailbending* 12.7 9.4 14.0 18.7 4.1 1.8 1.6 1.2 0.4 0.2 1.2 1.4 4.8 3.9 6.4 11.4 1.4 0.9 1.5 3.2 0.8 0.6 1.2 2.6 0.6 0.5 0.9 1.8 a c b a a b b b bc e ab a bc c b a b b b a b b b a b b a a i F r e q u e n c y p e r p e n p e r 5 o b s e r v a t i o n s (2.5 h). *P < 0.05; **P ,~ 0.01. U n d e r e a c h c o l u m n , m e a n s t h a t are f o l l o w e d b y t h e same l e t t e r are n o t significantly different. Means t h a t are f o l l o w e d b y d i f f e r e n t letters are s i g n i f i c a n t l y d i f f e r e n t b y D u n c a n ' s m u l t i p l e r a n g e tests. 377 Orthogonal comparisons showed t h a t males penned with females of their own line waltzed significantly m o r e oft en (5.2) than those p e n n e d with females o f the ot her line (3.6). Similar interactions were n o t significant for o t h e r c o m p o n e n t s of mating behaviour. Fertility and hatchability Eggs f r o m pens where bot h males and females were from the MM line had higher fertility than those collected from pens where the MM birds were crossed with 1-420 ones. Matings of 1-420 males with 1-420 females resulted in the lowest fertility (Table IV). Hatchability of eggs from t he crosses (MM X 1-420 and reciprocal) (88.2%) was significantly (P < ( 0 . 0 1 ) higher than eggs from line matings (MM × MM and 1-420 × 1-420) (78.2%), probabl y because o f heterosis. TABLE IV Fertility and hatchability of eggs collected during the study Mating types No. eggs set Fertility (%) Hatchability~ (%) MM X MM MM X 1-420 1-420 X MM 1-420 X 1-420 217 176 207 184 91.7 a 69.9 b 67.1 b 34.2 c 77.8 85.2 90.6 80.0 ~Percentage hatchability of fertile eggs. Under the fertility column, means that are followed by the same letters are not significantly different. Means that are followed by different letters are significantly different (P < 0.01) by x 2 tests. DISCUSSION Male libido is one of the m or e i m p o r t a n t factors affecting mating f r e q u e n c y in chickens (Craig et al., 1977) and turkeys (Carte and Leighton, 1969). Males with low libido mated less f r e q u e n t l y and fertilized fewer females (Wood-Gush and Osborne, 1956), while showing normal semen characteristics (McDaniel and Craig, 1959). These observations are consistent with those o f Parker (1961) and Soller et al. (1965) t hat the decreased fertility of Cornish males as compared t o that of other breeds is n o t due to differences in the fertilizing capacity of their semen, but because of their failure t o mate with as m a n y hens. F u r t h e r m o r e , the genetic correlation between behavioural and semen traits is low (McDaniel and Craig, 1959; Siegel, 1972). These and o t h e r studies, however, centered on the sexual behaviour of the male and its relationship to fertility. Although studies of female mating behaviour in chickens have not been extensive ( R a ppaport and Soller, 1966; Fischer, 378 1975), our data indicate that the problem of low fertility through natural mating in the 1-420 line was mostly due to a low mating frequency in the female as well as in the males. In this particular case, male and female behaviour seem to contribute equally to fertility (Table IV). Several studies indicated that the fertility is significantly higher with afternoon rather than morning inseminations (Moore and Byerly, 1942; Malmstrom, 1943; Parker, 1945; Bornstein et al., 1960; Johnston and Parker, 1970; Christensen and Johnston, 1975). Moreover, semen volume in roosters has been d o c u m e n t e d to vary diurnally, with highest volumes obtained in the afternoon (Lake and Wood-Gush, 1956). There were significantly fewer successful mounting attempts on 1-420 than on MM females in the afternoon, y e t the difference was not significant in the morning. Thus, the fewer copulations by 1-420 females during the optimum period might contribute to their lowered fertility. The first c o m p o n e n t of the signal-response sequence of mating behaviour in chickens, as outlined by Guhl and Fischer (1969), is the courting of the female by the male. The female either crouches or attempts to avoid the advance of the male. Crouching or failure to avoid may be followed by mounting, treading and tail-bending in that order. Either the male or the female may terminate the process at any stage during the sequence. Divergent selection for high and low frequencies of completed matings (CNCM) in male chickens (Cook and Siegel, 1974; Dunnington and Siegel, 1983; Bernon and Siegel, 1983) suggests that gene loci affecting the frequency of "courts" (includes approaches and waltzes (Guhl, 1961)), " m o u n t s " , "treads" and CNCM (i.e. tail-bending) show evidence for additive genetic variation, because these "traits" responded to selection. On the other hand, there was considerable heterosis for these traits when lines selected for mating frequency were crossed (Bernon and Siegel, 1983), indicating a significant amount of non-additive genetic variation in these loci also. When mating efficiency traits (ratios of CNCMs/courts, mounts/courts and CNCMs/mounts) were analyzed, non-additive genetic variation was found only in CNCMs/courts and mounts/courts but not in CNCMs/mounts. Dunnington and Siegel (1983) also reported high genetic correlations (0.82 and 0.83 in their high and low frequency mating lines, respectively) between CNCMs and mounts. The implication here is that, regardless of the male's genotype, once a m o u n t occurs there is a high probability that the mating will be completed. Based on these findings, one would expect inbreeding depression for traits associated with a significant amount of non-additive genetic variation (Falconer, 1960), but not in CNCMs/mounts where no non-additive genetic variation was found. In comparing our inbred (I-420) and the random-bred (MM) populations, we found that 1-420 males showed significantly fewer approaches, waltzes, treads and tail-bends -- observations consistent with expectation. On the other hand, there was no inbreeding depression in mounting attempts and successful mounting attempts. Furthermore, 1-420 379 males completed significantly fewer matings per successful m o u n t than MM males, indicating inbreeding depression in CNCMs/mounts. Although these observations m a y seem to be contrary to expectations, it should be remembered that the I 4 2 0 and MM lines did not originate from a c o m m o n foundation population, and the initial gene frequency for these traits may be different in the two populations. Moreover, observations in the different experiments were made under very different conditions, and traits, although under the same labels, may not be comparable. Males may react differently if they only have limited access to the females (as in the selection experiments) compared to being kept with females all the time. Siegel (1959) f o u n d significant differences in male mating behaviour among four inbred lines of White P l y m o u t h Rock and concluded t h a t the frequency of mating behaviour of the inbreds was not always lower than that of heterogeneous stocks. Courtship behaviour (waltzing) in male chickens is both agonistic and sexual (Krujit, 1966; Guhl and Fischer, 1969). In our study, males waltzed significantly more frequently in the morning when females were least willing to crouch, suggesting that these waltzes may have large aggressive components. Males also waltzed significantly more frequently when they were kept with females of their own line rather than with females of the other line. 1-420 and MM males were probably sexually imprinted on females of their own type and this difference in frequency of waltzing revealed the sexual nature of waltzes. Cook et al. (1972) indicated that the inheritance of waltzing is different from that for frequency of completed matings. Inbreeding appeared to have increased the threshold for crouching in 1-420 females. Although aggressiveness of the females also affects the threshold for the crouching response, we do not know if the same set of loci that affect mating behaviour in males also affect female mating behaviour. It is possible that although mating behaviour of males and females depends on an association of hormones and threshold levels (Van Krey et al., 1977), the components of the relationship in the sexes are different (Nalbandov, 1964). After 23 generations of selection for male mating behaviour, Dunnington and Siegel (1983) found no consistent differences among lines for female mating behaviour. Thus, the genotype that affects the hormone-threshold relationship in one sex may not necessarily affect such a relationship in the same manner in the opposite sex (Cook and Siegel, 1972). In summary, our results regarding the mating behaviour of the 1-420 inbred chickens which had low mating frequency is consistent with previous findings on sexual behaviour of domestic chickens. Furthermore, our experiment also revealed the intricate interaction between male and female behaviour in affecting fertility. ACKNOWLEDGEMENTS This study was conducted while K.M. Cheng was a research specialist at the J.F. Bell Museum of Natural History, University of Minnesota. This 380 position was supported by a grant from the National Science Foundation ( B N S - 7 9 4 6 9 2 ) . W e w o u l d l i k e t o t h a n k P.B. S i e g e l a n d D . M . S h a c k l e t o n f o r c r i t i c a l c o m m e n t s o n a n e a r l i e r d r a f t o f t h i s m a n u s c r i p t . T h i s is c o n t r i b u t i o n N o . 13 6 2 0 o f t h e S c i e n t i f i c J o u r n a l S e r i e s o f t h e M i n n e s o t a A g r i c u l t u r a l Experiment Station. REFERENCES Bemon, D.E. and Siegel, P.B., 1983. 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