Maternal Olfaction Differentially Modulates Oxytocin and Prolactin Release during Suckling in Goats

Hormones and Behavior 42, 232–244 (2002) doi:10.1006/hbeh.2002.1812

Maternal Olfaction Differentially Modulates Oxytocin and Prolactin Release during Suckling in Goats Horacio Hernandez,* Norma Serafin,* Ange´lica M. Terrazas,* Pierre Guy Marnet,† Guy Kann,‡ Jose´ Alberto Delgadillo,§ and Pascal Poindron* ,1 *Centro de Neurobiologı´a, Universidad Nacional Auto´noma de Me´xico, AP 1-1141, Juriquilla, Quere´taro 76001 Qro., Me´xico; †Unite´ mixte INRA/ENSAR de Recherches sur la Production du Lait, INRA, 65, rue de Saint-Brieuc, 35042 Rennes Cedex, France; ‡Unite´ de Recherches sur l’Endocrinologie du Placenta et de la Pe´rinatalite´, Laboratoire de Biologie Cellulaire et Mole´culaire, INRA, 78352 Jouy en Josas Cedex, France; and §Departamento de Ciencias Me´dico Veterinarias, Universidad Auto´noma Agraria Antonio Narro, Carretera a Santa Fe y Perife´rico, AP 940, Torreo´n Coahuila, Me´xico Received January 21, 2002; accepted March 11, 2002

In postparturient goats, olfactory recognition of the young allows the establishment of a selective bond between the mother and her kids. Once this bond is formed, the mother rejects alien young that attempt to suckle. We tested whether the development of the maternal selective bond in goats modulates prolactin (PRL) and oxytocin (OT) release in response to suckling. On day 37 of lactation, serial blood samples were taken during nursing of the mother’s own or alien kid(s) in 10 intact/selective goats and in 10 goats rendered anosmic/nonselective through prepartum peripheral ZnSO 4 irrigation. Spontaneous nursing behavior was also studied weekly from day 7 to 30 of lactation, at which time milk production was measured. Maternal selectivity had no effect on PRL release, in contrast to OT release, which was significantly affected by this factor. Intact mothers released OT only when nursing their own kids, but not with aliens, while anosmic/nonselective dams showed an increase in OT levels regardless of the identity of the kids. In addition to these effects on maternal selectivity, the amplitude of the response of both hormones was lower in anosmic mothers than in intact mothers. Finally, nursing behavior and milk production were not significantly affected by anosmia. We conclude that maternal selective behavior in goats, which relies on the individual olfactory signature of the kid, modulates

1 To whom correspondence and reprint requests should be addressed at present address: UMR 6073, Physiologie de la Reproduction et des Comportements, INRA, 37380 Nouzilly, France. Fax: 52-4422-2381046. E-mail: [email protected].

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the OT, but not the PRL, response to suckling. In addition, perception of the smell of the young appears to have a general facilitatory effect, independent of the kid’s identity, on the release of both hormones. © 2002 Elsevier Science (USA)

Key Words: goat; maternal behavior; suckling; prolactin; oxytocin; olfaction; anosmia; selectivity; lactation.

Mammals show different patterns of mother–young interactions in relation to the developmental status of the neonate (Gubernick, 1981; Gonzalez-Mariscal and Poindron, 2002). Thus, in altricial species (most rodents and lagomorphs), the mother builds a nest in which she gives birth to offspring that are not well developed. Mother–young interactions occur within the nest and in general do not depend strongly on mutual recognition. On the other hand, precocial species (e.g., ungulates) give birth to well-developed offspring, which stand and walk as early as 30 min after birth and start to suckle within 1 h. Moreover, an exclusive and enduring bond between the mother and her offspring is rapidly established shortly after parturition. Thereafter, the mother accepts only her own young at the udder and actively rejects any alien that attempts to suckle (this aspect of maternal behavior is referred to as maternal selectivity; sheep, Bouissou, 1968; goats, Hersher, Richmond, and Moore, 1963; cattle, Hudson and Mullord, 1977). It is also well es-

0018-506X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

tablished that in sheep and goats, this maternal selectivity depends on the individual olfactory signature of the offspring (Poindron and Le Neindre, 1980; Porter, Le´ vy, Poindron, Litterio, Schaal, and Beyer, 1991; Porter, Romeyer, Le´ vy, Krehbiel, and Nowak, 1994; Romeyer, Porter, Le´ vy, Nowak, Orgeur, and Poindron, 1993). In mothers of altricial species, which do not develop selective nursing, it is well established that the stimulus caused by suckling of the pups induces the release of prolactin (PRL) and oxytocin (OT) into the peripheral blood (Grosvenor, Shyr, Goodman, and Mena, 1986; Fuchs, Cubile, Dawood, and Stener-Jørgensen, 1984). These hormones stimulate the synthesis and ejection of milk from the mammary glands. Nevertheless, in the lactating rat, PRL release is not exclusively caused by the suckling stimulus of the offspring, since perception of the pups’ olfactory cues alone can elicit PRL release from the maternal pituitary gland similar to that induced by suckling (Grosvenor, 1965; Mena and Grosvenor, 1971). Also, in certain circumstances, auditory cues from the pups may be important for PRL release (Terkel, Damasa, and Sawyer, 1979; Stern and Siegel, 1978; Stern, Thomas, Rabii, and Barfield, 1984). Finally, toward the final phase of lactation, it has also been reported that mother rats may release PRL in response to exteroceptive stimulation, from both their own pups and alien pups of other lactating mothers (Mena and Grosvenor, 1972). While the effect of suckling on PRL and OT release is also well documented in species that establish an exclusive bond with their litter (goats: Buttle, Forsyth, and Knaggs, 1972; McNeilly, 1972, sheep: Kann, Habert, Meusnier, and Ryniewicz, 1977; cattle: Akers and Lefcourt, 1982), there is little information concerning the relationships between the presence of maternal selective behavior and the endocrinology of lactation. Indications that exteroceptive stimuli can induce hormonal release in lactating females have also been reported in these species. In goats, Hart (1974) suggested that PRL release can be induced not only by the tactile stimulation of the teats due to suckling, but also due to a conditioned response associated with the events which take place just before milking or in response to a metabolic stimulus. Nevertheless, in a later study Hart and Linzell (1977) showed that stimuli associated with the milking routine did not appear to induce a conditioned released of PRL. Also, mechanical stimulation of the teat by milking or suckling did not induce PRL or OT release in females in which the udder had been denervated (Kann, 1980). In addition, McNeilly (1972) found that few goats (2 of 12) responded with a

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conditioned release of oxytocin to exteroceptive cues from kids. On the other hand, there are some indications that maternal selectivity might influence PRL or OT release at the time of suckling. For instance, in cattle, Perez, Jimenez de Perez, Poindron, Le Neindre, and Ravault (1985) showed that the PRL response to machine milking was significantly lower than that induced by nursing of the mother’s own calf and that suckling by an alien calf did not lead to any significant response. Also, Silveira, Spoon, Ryan, and Williams (1993) found that the proportion of cows experiencing OT release following controlled suckling was greater in mothers nursing their own calf than in dams forced to nurse an alien calf. Goats, like sheep and cattle, also develop a selective maternal behavior within the first hours postpartum on the basis of olfactory cues from the kids, and mothers made anosmic before parturition do not develop maternal selectivity (Romeyer, Poindron, and Orgeur, 1994; Herna´ndez, 2001). Nevertheless, whether maternal selectivity modulates PRL and OT release in response to suckling depending on the identity of the young has not been investigated thus far in this species. Also, while maternal olfaction appears to be of major importance for the establishment of the maternal bond, it is not known whether prepartum anosmia may result in detrimental effects on nursing behavior, as previously reported in sheep (Poindron, 1976a). The purposes of the present study were therefore (1) to determine whether maternal selectivity modulates the PRL and OT response to suckling, depending on the identity of the kid, in selective (intact) and nonselective (anosmic) lactating goats, and (2) to assess any possible effect of anosmia on nursing behavior.

METHODS Animals The subjects were 20 Mexican dairy crossbred (mainly from Alpine and Saanen breeds) goats, 10 of which remained intact (selective group) and 10 of which were made anosmic 1 month before kidding (nonselective group). In each group, 7 goats reared twins, and 3 reared a single kid. In the intact group, 6 goats were primiparous, and 4 were multiparous, while the anosmic group consisted of 5 primiparous and 5 multiparous mothers.

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Anosmia Procedure In anosmic goats, the olfactory mucosa was destroyed by two successive intranasal irrigations with a solution of zinc sulfate (1.5%) and procaine chlorhydrate (3%) under general anesthesia (Romeyer et al., 1994) at a 1 week interval. The first treatment was performed 1 month before the expected date of parturition. Olfactory deficits were confirmed 24 h postpartum and on day 30 of lactation with a 2-min behavioral feed preference test (Poindron, 1976b). Each female was tested individually, giving her the choice between two buckets, both containing concentrate pellets, but one with the upper part of its walls coated with dog excrement that was not in contact with the food. All animals treated were found to be anosmic according to this test (i.e., consumed food associated with the odor of dog excrement for more then 10 s, while intact animals did not consume any) during the two postpartum periods. Maintenance Conditions and Postpartum Selectivity Test Animals were fed lucerne hay and concentrate (Generaleche, with 18% crude protein, Purina), with mineral supplements and water available ad libitum. Rations were adjusted according to the goats’ physiological status (stage of pregnancy and lactation), as well as for litter size (NRC, 1981). Before parturition, animals were kept under intensive management at a density of 5 m 2/animal. Immediately after parturition, mothers and their kid(s) were placed in contiguous individual pens (2 ⫻ 2 m) provided with shade, where they remained until the end of the study. To ensure that anosmia was associated with the absence of selectivity, all mothers were tested for their response to an alien kid at 24 h postpartum. To this end, each of the mothers in both groups was separated from her kid(s) and placed in a different testing pen (2 ⫻ 2 m) for 5 min. A kid (own or alien, in a balanced order in each group) was then introduced and behavior of the female was observed for 2 min. The behaviors registered were as follows: number of low- and high-pitched bleats, acceptance at the udder, duration of nursing, rejection at the udder, head butts, and threats. Nursing Behavior The first monitoring of spontaneous nursing began on day 7 after parturition and was followed by three

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further periods of observation at intervals of 7 ⫾ 1 days, each session lasting 2 h/mother. The onset of observations varied from 08:00 to 10:00 h and was randomized between groups. When observing mothers with twins, one observer followed only one mother at any one time. In the case of the mothers with single kids, two mothers were followed at the same time. The observers were located in a passage contiguous to the individual pens. In the present article, we use the terminology proposed by Hall, Hudson, and Brake (1986), where the term nursing refers to the activity of the mother and suckling to the activity of the young. Identity of the kid, duration of suckling for each kid, and rejections were recorded on paper. Rejection rate was calculated by dividing the number of rejections by the sum of the number of successful nursing episodes plus the number of rejections at the udder. The nursing parameters were defined in accordance with the criteria from Delgadillo, Poindron, Krehbiel, Duarte, and Rosales (1997). A nursing episode was defined as the period during which a dam nursed without interruption for more than 3 s, regardless of the number of kids that suckled. Rejection was defined as any attempt by a kid to suckle that lasted less than 3 s after its beginning and was interrupted by the dam either moving away or responding aggressively toward the kid. The behavioral parameters assessed for nursing (mothers) and suckling (kids) were their frequency, mean, and total duration/2 h, as well as the rejection rate (%) for nursing attempts. Hormonal Response to Suckling on Day 37 of Lactation The serial blood samples to study the PRL and OT responses to suckling were obtained in the individual pen in which the mother was normally staying with her young. A catheter was inserted into the jugular vein of each female at 24 h before sampling and fixed in the vein of the animal by sewing it on the skin under local anesthesia (2% Xylocaine). Heparin was used as an anticoagulant to keep the catheter functional. At the time of sampling and during the time the young were present in the pen, the mothers were gently restrained, always by the same person to minimize stress due to handling. Also, when the young were introduced in the pen and at the beginning of suckling, the mother’s head was positioned so that her nose was less than 2 cm away from the body of the young, and she was allowed to smell them during the entire time they were present in the pen. Thus, the sampling conditions were the same in all cases, re-

Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

gardless of the group and of the identity of the kids. All blood samples were obtained in the morning between 08:00 and 11:00 h. On each occasion, 2 h before the sampling procedure, the kids (own or alien) to be used for suckling were removed from their mother and placed in a distant pen out of sight of the mother. However, acoustical isolation was not possible and the bleats emitted by the kids due to separation could be heard by the dam. Samplings with the own and the alien kids were made on 2 consecutive days and the order of the type of kid was balanced in both groups. The first two samples were taken at 3 and 2 min before the onset of suckling, this latter time being considered as time 0. After the second sample was obtained, the kids were held in front of the female for 2 min, during which time she could see, hear, and smell the kid(s) but not nurse. A third sample was taken after the first minute of kid exposure and a fourth at the end of this period, 1 min later. The kids were then immediately released and allowed to suckle. Additional blood samples were taken at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 10.0, 15.0, 20.0, and 30.0 min after the onset of teat stimulation, which consisted of 2 min of suckling by the own kid or an alien kid of the same age. In the case of mothers with twins, both kids were used for the udder stimulation and two alien kids for the alien condition. Five milliliters of blood was collected for each hormone in tubes kept on ice. After centrifugation at 4000 rpm at 4°C, plasma was stored at ⫺20°C until hormonal assay. Hormonal Assays Plasma PRL concentrations were determined by RIA in accordance with the method described by Kann (1971). The limit of detection was 1.24 ng/ml ⫺1, and the inter- and intra-assay coefficients of variation were 4.5 and 12.6% for 1.36 ng/ml ⫺1, respectively. Plasma OT levels were determined by enzyme immunoassay (Marnet, Volland, Pradelles, Grassi, and Beaufils, 1994). The limit of detection was 1.5 pg/ml ⫺1, and the inter- and intra-assay coefficients of variation were 13 and 8.6% for 4 pg/ml ⫺1, respectively. Milk Production at 1 Month of Lactation In all mothers, milk production was assessed at 1 month of lactation, based upon differential body weight of the kids before and after suckling over a period of 24 h (Ricordeau, Boccard, and Denamur, 1960). The day before initiating the measurements, a first emptying of the udder was performed at 18:00 h,

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followed by three controlled sucklings of 4 min each on the following day at 08:00, 13:00, and 18:00 h. Moreover, after each controlled suckling, 2 IU of exogenous OT was injected into the jugular vein, followed by hand milking to extract any residual milk. The weight of this residual milk was added to that of the corresponding body weight measurement. Statistical Analysis The behaviors recorded in the selectivity tests were analyzed using the Mann–Whitney test for independent data and the Wilcoxon test for two related samples. Nursing and suckling variables from spontaneous maternal behavior were transformed to logarithms to approximate a normal distribution and analyzed with a two-way repeated-measures ANOVA. Hormonal data were analyzed using twoway MANOVA (group as between-factor and time of sampling and identity of the kids as within-factors) to detect general effects. Moreover, the effect of kid identity within each group was analyzed by two trial factor MANOVA. Likewise, the group effect was analyzed for each type of kid (own or alien) using a two-way MANOVA (group as between-factor and time of sampling as within-factor). For OT levels, the data were first approximated to a normal distribution through logarithm transformation (Moore and McCabe, 1989). Milk production on day 30 of lactation was compared between groups with a t test. All statistical analyses were performed using the package Systat 7.0 (SPSS, Chicago). Results are given as means ⫾ standard error (SEM). Ethical Note Animal care and experimental treatments complied with Guidelines A37801 of the French Ministry of Agriculture for animal experimentation, which were also used in a former collaborative study with French scientists concerning the effects of anosmia on mother– young recognition in sheep in the frame of a collaborative work (Ferreira, Terrazas, Poindron, Nowak, Orgeur, and Le´ vy, 2000). In addition, anosmia neither altered the growth rate of the goats during the last 2 months of pregnancy nor induced abortions or any observable changes in the general behavior of the females, which is consistent with what has been previously reported in sheep (Herna´ ndez, Serafin, Va´ squez, Delgadillo, and Poindron, 2001). Furthermore, no differences were detected between groups in the birth weight of the kids. None of the selectivity tests re-

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TABLE 1 Behavioral Responses (Mean ⫾ SEM) of Intact and Anosmic Goats Toward Own and Alien Kid in a 2-min Test of Maternal Acceptance at the Udder Performed at 24 h Postpartum Intact goats (n ⫽ 10) Behavior

Own

Udder acceptance (frequency) Nursing time (s) Low-pitch bleats (frequency) High-pitch bleats (frequency) Aggressive behavior (frequency)

1.8 ⫾ 0.4 29.0 ⫾ 11.7 6.7 ⫾ 1.7 2.8 ⫾ 2.2 0

Anosmic goats (n ⫽ 10)

P

Alien

* * n.s. * *

0.1 ⫾ 0.1 0.7 ⫾ 0.7 6.3 ⫾ 2.6 a 14.0 ⫾ 5.4 a 2.3 ⫾ 1.3 a a

Own

P

Alien

1.5 ⫾ 0.3 29.0 ⫾ 10.3 7.2 ⫾ 2.1 0.2 ⫾ .02 0

n.s. n.s. * n.s. n.s.

0.7 ⫾ 0.2 b 15.0 ⫾ 10.3 11.3 ⫾ 1.8 b 2.3 ⫾ 2.1 b 0.4 ⫾ 0.4 b

Note. P Comparison between own and alien (*P ⬍ 0.05; n.s., P ⬎ 0.05; Wilcoxon test). a,b In each row, different letters indicate differences in behaviors toward the alien kid between intact and anosmic goats (Mann–Whitney test).

sulted in permanent disruption of the relationship between a goat and her kids or in injury to the kids. Kids were readily accepted by their mother and allowed to suckle. There were also no consequences of these treatments on growth rate of the kids until weaning.

both groups. An effect of time was also found for the total duration of nursing in anosmic mothers (Table 2). On the other hand, no significant change over time was found for any of the variables concerning the kids (Table 2).

RESULTS

Hormonal Response to Suckling

Maternal Behavior Selectivity Test at 24 h Postpartum In intact mothers, all behaviors except maternal bleats differed significantly between the own and alien kid conditions (Wilcoxon test, P ⬍ 0.05 in all cases). On the contrary, this was not the case in anosmic mothers. Furthermore, the behaviors of the mothers toward their own kid did not differ significantly between intact and anosmic dams, whereas differences were observed in their behavior toward the alien kids (Mann–Whitney test, P ⱕ 0.05; Table 1). Altogether, these results show that anosmia was actually associated with the absence of selective behavior in this group. Spontaneous Behavior of the Mothers and Their Kids Results of the observations of spontaneous maternal nursing behavior and suckling behavior by kids are presented in Table 2. No significant differences were found in the nursing behavior parameters between intact and anosmic goats at any time during the study (P ⱖ 0.87, for all variables; Table 2). On the other hand, a significant effect of time (but no interaction between time and group) was found regarding the frequency of nursing episodes, which decreased in

A preliminary comparison (MANOVA) of hormonal levels obtained in multiparous and primiparous goats within each group did not reveal any effect of the previous number of parturitions, neither for PRL nor OT (P ⬎ 0.20). Similarly, no effect of the number of kids raised by the dam was found for either hormone (P ⬎ 0.19). Finally, the presence of the young during the 2 min preceding suckling had no effect on prenursing levels of PRL or OT. Therefore, the basal levels of these hormones were defined as the mean of the three (PRL) or four (OT) prestimulation values. PRL. Results for this hormone are presented in Fig. 1. In intact mothers, the PRL concentrations increased from basal levels of 58 ⫾ 13 ng/ml and 72 ⫾ 12 ng/ml to maximum values of 112 ⫾ 18 ng/ml and 116 ⫾ 9 ng/ml for own and alien kids, respectively. In anosmic dams, the corresponding values were 77 ⫾ 12 ng/ml and 66 ⫾ 11 ng/ml for basal levels and 93 ⫾ 11 ng/ml and 85 ⫾ 11 ng/ml for maximum levels. The general analysis revealed a main effect of the time of sampling on PRL concentration (P ⬍ 0.001; Fig. 1), but no effect of group or identity of the kid. On the other hand, a significant interaction was found between group and time of sampling (P ⫽ 0.014; Fig. 1), indicating some effect of anosmia on the amplitude of release of this hormone. In intact dams, PRL levels were increased both during suckling by the own kids

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Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

TABLE 2 Characteristics of Spontaneous Nursing (Mothers) and Suckling (Kids) Behaviors (mean ⫾ SEM) in Intact and Anosmic Crossbred Dairy Goats during the First Month of Lactation Time postpartum

Mothers Intact group Number of nursing episodes/dam/2 h Total duration of nursing episodes/dam (min) Mean duration of a nursing episode/dam (s) Rejection rate at nursing (%) Anosmic group Number of nursing episodes/dam/2 h Total duration of nursing episodes/dam (min) Mean duration of a nursing episode/dam (s) Rejection rate at nursing (%) Kids Intact group Total number of suckling episodes/kid/2 h Total duration of suckling/kid (min) Mean duration of a suckling period/kid (s) Anosmic group Total number of suckling episodes/kid/2 h Total duration of suckling/kid (min) Mean duration of a suckling period/kid (s)

Week 1

Week 2

Week 3

Week 4

Evolution with time

8.5 ⫾ 2.7 3.3 ⫾ 0.9 25.7 ⫾ 3.5 2.3 ⫾ 1.2

4.7 ⫾ 0.8 2.1 ⫾ 0.7 24.2 ⫾ 5.4 28.9 ⫾ 10.2

2.8 ⫾ 0.5 1.4 ⫾ 0.3 30.1 ⫾ 5.6 42.6 ⫾ 10.5

3.2 ⫾ 0.6 2.2 ⫾ 0.9 41.0 ⫾ 16.7 43.4 ⫾ 10.6

P ⬍ 0.05 n.s. n.s. P ⬍ 0.05

8.1 ⫾ 2.2 4.8 ⫾ 1.6 32.1 ⫾ 5.7 2.9 ⫾ 2.1

4.0 ⫾ 0.8 2.2 ⫾ 0.5 28.6 ⫾ 4.3 37.5 ⫾ 7.9

3.5 ⫾ 0.5 1.5 ⫾ 0.2 26.2 ⫾ 1.7 31.1 ⫾ 8.8

4.0 ⫾ 1.0 1.5 ⫾ 0.3 25.0 ⫾ 4.3 31.0 ⫾ 7.3

P ⬍ 0.05 P ⬍ 0.05 n.s. P ⬍ 0.05

5.4 ⫾ 1.5 2.1 ⫾ 0.5 24.4 ⫾ 3.1

3.5 ⫾ 0.7 1.6 ⫾ 0.4 22.0 ⫾ 10.5

2.7 ⫾ 0.5 1.2 ⫾ 0.2 25.7 ⫾ 3.5

3.3 ⫾ 0.4 2.0 ⫾ 0.5 39.0 ⫾ 10.6

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

6.1 ⫾ 1.4 3.4 ⫾ 0.9 28.3 ⫾ 4.8

3.4 ⫾ 0.5 1.7 ⫾ 0.3 29.3 ⫾ 2.9

3.6 ⫾ 0.4 1.5 ⫾ 0.2 25.6 ⫾ 1.8

4.1 ⫾ 0.7 1.6 ⫾ 0.2 26.0 ⫾ 3.2

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

Note. No significant differences were found in the nursing behavior parameters between intact and anosmic goats at any time during the study (P ⱖ 0.87, for all variables).

and during suckling by the alien kids, whereas in anosmic mothers, suckling by the kid(s) induced a smaller response, which was significant only in the case of the alien kid(s). OT. Results for this hormone are presented in Fig. 2. In the intact group, OT basal levels were 33 ⫾ 14 pg/ml and 16 ⫾ 3 pg/ml for the own and the alien kids, respectively. In the anosmic group, basal levels were 17 ⫾ 2 pg/ml and 22 ⫾ 8 pg/ml, and peak levels were 49 ⫾ 7 pg/ml and 49 ⫾ 12 pg/ml, for own and alien kids, respectively. While the prestimulation basal levels of this hormone in the selective group appeared to be higher with the own kid than with the alien, this was not significant (P ⫽ 0.14, Wilcoxon) and it is important to stress that this difference was due to one single goat, which presented a high OT level (143 pg/ml) before the introduction of her own kid. Furthermore, excluding this animal did not change the significance of any of the analyses. The MANOVA revealed significant main effects of identity of the kid (P ⫽ 0.02) and time (P ⬍ 0.001), as well as a significant interaction between time of sampling and identity of the kid (P ⬍ 0.001) and a tendency for an interaction between group, time, and

identity of the kid (P ⫽ 0.06). Indeed, a significant effect of anosmia on the amplitude of release was found in the case of suckling by the own kid: the OT response was different depending on the identity of the kids in intact/selective mothers, while in anosmic/nonselective dams this was not the case (Fig. 2). In intact mothers the response induced by the own kids consisted of an increase in OT basal levels 1 min after the onset of nursing. By contrast, these intact dams showed no significant changes in OT levels in response to suckling by alien kids. In anosmic mothers, on the other hand, a main effect of identity of the kids was not found (P ⬎ 0.1), whereas there was a clear response to suckling by the alien kid (P ⬍ 0.001; Fig. 2). Milk Production 30 days Postpartum The quantity of milk produced during a 24-h period by the mothers did not differ between intact and anosmic goats (1.7 ⫾ 0.2 kg and 1.9 ⫾ 0.3 kg, respectively; ANOVA, P ⫽ 0.99). On the other hand, a significant difference was found between mothers of a single kid and mothers of twins (1.29 ⫾ 0.20 kg and 2.01 ⫾ 0.20

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FIG. 1. Temporal pattern of mean (⫾SEM) PRL plasma levels in response to suckling by own kids (top) or alien kids (bottom) in intact/selective and anosmic/nonselective goats at 37 days of lactation. Taking into account all data, the general MANOVA revealed a significant interaction between group and time of sampling (P ⫽ 0.014).

kg, P ⫽ 0.05), but no interaction between group and number of kids.

DISCUSSION Two main conclusions can be drawn from our results. First, the study carried out in intact mother goats indicates that maternal selectivity does not exert the same control on PRL and OT release induced by suckling. While the identity of the young that suckles has no influence on prolactin release, it is of major importance for the release of oxytocin. This is further confirmed by the fact that prepartum anosmia in the mother results in a similar and significant increase of

OT levels when the mother is suckled by her own kid or by an alien kid, whereas the release of prolactin remains similar for the two types of kids. The second point revealed by our study is that the absence of olfactory perception in the mother is associated with globally lower levels of both PRL and OT in response to suckling of the own young, while it has no effect on milk production or global nursing activity. As a whole, it appears therefore that the exteroceptive olfactory stimuli from the kids have a differential and dual modulatory action on the secretion of these hormones in response to suckling: on the one hand, the perception of these olfactory cues has an overall facilitatory action on the release of both hormones when the mother is nursing her own young, and, on the

Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

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FIG. 2. Temporal pattern of mean (⫾SEM) OT plasma levels in response to suckling by own kids (top) or alien kids (bottom) in intact/selective and anosmic/nonselective goats at 37 days of lactation. Taking into account all data, the general MANOVA revealed a tendency for an interaction between group, time of sampling, and identity of the kid (P ⫽ 0.06).

other hand, the perception of the individual smell of an alien kid totally blocks the release of OT. To our knowledge, this is the first time that such an interaction between exteroceptive and proprioceptive stimuli on PRL and OT secretion has been reported in goats and that the effects of maternal anosmia on these parameters have been investigated in this species. Our present results are also in agreement with the observations made previously in dairy Lacaune ewes submitted to a mixed system of suckling and milking (Marnet and Negrao, 2000). These authors found that milking in maternal ewes was not followed by OT

release, while cortisol release and prolactin release were maintained. Our results are also congruent with the absence of conditioned OT release to milking found in this species by Marnet, Negrao, and Labussie`re (1998). Effects of Maternal Selectivity on Hormonal Release Regarding OT, our results are consistent with the idea proposed for other species that the release of this hormone can be affected by the expression of maternal

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exclusive acceptance of the young at the udder. Thus, in cattle, which also show selective nursing (Hudson and Mullord, 1977; Le Neindre and Garel, 1979), the proportion of dams showing OT release following suckling by the own calf on day 4 of lactation was higher than that of dams forced to nurse an alien young (Silveira et al., 1993). These results are opposed to those found in the rat, in which lactating mothers will nurse alien pups even though they are able to discriminate between them and their own offspring (Beach and Jaynes, 1956). For example, Grosvenor et al. (1986) found a significant increase in the peak plasma OT concentration following suckling by a litter of six hungry alien pups, even after the mother had nursed her own pups. This is further supported by the fact that in the present study, prepartum anosmia, which impedes the establishment of selective nursing in goats (Klopfer and Gamble, 1966; Romeyer et al., 1994), resulted in the release of OT in response to suckling by an alien kid, even though anosmic mothers are still able to discriminate their own young on the basis of nonolfactory cues (Gilling, 2002). OT release in anosmic mothers also indicates that the failure to observe OT release in the presence of an alien kid in intact mothers is not due to the absence of the individual odor of the own kid. If this were the case, OT response by anosmic dams would not have been observed in the presence of either type of kid. Taken together, our results indicate therefore that individual olfactory cues from the young, which are the basis for maternal selectivity, also modulate the release of OT at the time of nursing. It is important to stress, however, that the presence of exteroceptive cues by themselves did not promote the release of OT or PRL, contrary to what has been previously reported for PRL in the rat (Mena and Grosvenor, 1971; Terkel et al., 1979) and also in mastectomized cattle (Stevenson, Knoppel, Minton, Salfen, and Garverick, 1994). In addition, the modulation of suckling-induced release of PRL by maternal selectivity does not appear as consistent as for OT. In the present study, we failed to find any effect of the identity of the kid on this variable, which is consistent with results reported by Silveira et al. (1993) in cattle, but not with those of Perez et al. (1985), who found that suckling by an alien calf did not lead to any significant PRL response. General Effects of Anosmia on Hormonal Release The results obtained in anosmic mother goats suggest that the influence of olfactory cues from the young is not limited to their participation in maternal

Hernandez et al.

selectivity. While no positive effects of exteroceptive stimuli were observed in the absence of suckling, anosmia resulted in a significantly smaller release of PRL in response to the suckling by the own and the alien young and also in a lower OT response to suckling by the own young. It appears therefore that in addition to the selectivity-dependent effect of olfactory cues on OT release, olfactory perception has a general positive effect on PRL and OT release induced by suckling. Such an effect of anosmia is similar to that reported in the rat for PRL, although in this latter species the presence of olfactory cues from the pups was sufficient to induce hormonal release in intact dams (Mena and Grosvenor, 1971). In addition, despite the fact that in the present study exteroceptive stimuli from the young were unable to induce a release of OT independently of suckling, this does not exclude the possibility that such a release may occur. Thus, McNeilly (1972) found that some goats (2 of 12 goats in his study) showed an OT release before suckling, due to exteroceptive sensory stimulation from the kids. Similarly, in sheep, OT release was induced by exteroceptive stimulation emanating from the lamb (Fuchs, Ayromlooi, and Rasmussen, 1987). One possible reason for the differences between these results and those of the present work may be the day of lactation on which the studies were performed. Thus, in the study by Fuchs et al. (1987) the samples were obtained between days 1 and 15 postpartum versus between day 1 and 21 of lactation in the study of McNeilly (1972) and on day 37 postpartum in our study. In species such as rats and rabbits, it seems that the ability of either suckling or exteroceptive stimulation from the pups to modulate PRL release or lactation duration depends on the lactation stage of the mother and the age of the pups (Grosvenor, Shah, and Crowley, 1991; Mena, Clapp, and Martı´nez de la Escalera, 1990; Mena, Clapp, Aguayo, Morales, and Martı´nez de la Escalera, 1991). An alternative to the hypothesis of a direct involvement of olfaction in the modulation of PRL and OT release is that behavioral stress affects the response of these hormones to suckling in intact goats. Suckling by an alien kid may be quite a stressful event for an intact dam, thus resulting in the release of ␤-endorphin and inhibition of OT release, as well as in stress-induced PRL release. However, although the inhibitory influences of opiates on OT release are well established in the rat (Wakerley, 1994; Russell, Coombes, Leng, and Bicknell, 1993), their exact role in goats or ruminants in general is not clearly understood (Aurich, Dobrinski, Hoppen, and Grunert, 1993; Bruckmaier and

Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

Blum, 1996; Bruckmaier, Shams, and Blum, 1993; Gordon, Renfree, Short, and Clarke, 1987; Hashizume, Haglof, and Malven, 1994; Seckl and Lightman, 1987). Also, if stress was responsible for the PRL response observed in intact mothers nursing an alien kid, differential effects of anosmia should have been found between the responses to own vs alien young. Therefore, if some effect of stress cannot be excluded, our results appear more in concordance with a specific modulation of hormonal release by olfactory stimuli. The neurobiological mechanisms that control the differential effects of olfaction on the suckling-induced release of PRL and OT in goats are not known. Several studies in rats have provided evidence for a direct synaptic input from the main and accessory olfactory systems to supraoptic nuclei, which could be involved in the modulation of OT release by olfactory cues (Hatton and Yang, 1989; Smithson, Weiss, and Hatton, 1989; Smithson, Weiss, and Hatton, 1992). Also, it is interesting to note that some parallels can be made between our results and those concerning the electrophysiological responses found in the olfactory bulb of the postparturient sheep, a species in which selectivity of maternal behavior also depends on olfaction. While a large proportion (70%) of cells respond to the odor of any lamb, others (30%) respond preferentially to the odor of the ewe’s own lamb (Kendrick, Le´ vy, and Keverne, 1992). Moreover, when a dam established a selective bond with her lamb, its odor, but not that of aliens, produced an increase in the release of both glutamate and GABA in the olfactory bulbs. By contrast, both own and alien lamb odors produce an increase of acetylcholine and noradrenaline release, suggesting that these two latter neurotransmitters are important for general lamb odor identification, while glutamate and GABA are involved in the selective olfactory recognition of the lamb. Therefore, it is possible that the endocrine effects found in goats may depend on a similar differential activation of the first olfactory relay located in the olfactory bulbs. If this hypothesis is correct, it would also be congruent with the absence of differences in the hormonal responses of primiparous and multiparous mothers, since at the neurobiological level, the differences reported in the olfactory bulb disappear within a few hours postpartum (Le´ vy, Porter, Kendrick, Keverne, and Romeyer, 1996). Our results are also in agreement with those of Negrao, Marnet, and Kann (1998), who reported that the PRL response to milking by dairy sheep did not differ between the first and the second lactation in the same animals.

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Effects of Anosmia on Behavior and Milk Production In the light of the differences found between intact and anosmic mothers in their PRL and OT responses to suckling, it may seem surprising that milk production did not differ between the two groups. However, since neither PRL or OT responses were totally suppressed, it is likely that sufficient levels of both hormones were released for initial lactogenesis, as well as for maintenance of milk synthesis and udder evacuation. This is further supported by the fact that udder evacuation, an essential process for maintenance of milk production (Wilde and Peaker, 1990; Peaker and Wilde, 1996), does not appear to depend as strongly on OT release in the goat as it does in other species that have a smaller cisternal milk fraction (Marnet and McKusick, 2001). It is also consistent with the fact that anosmia did not significantly modify maternal nursing activity, contrary to the results reported earlier for sheep (Poindron, 1976a), in which nursing activity was decreased in anosmic mothers. A likely reason for this difference is the fact that anosmia was induced peripherally by irrigation of the olfactory mucosa with ZnSO 4 versus olfactory bulbectomy in the study of Poindron (1976a). This is also in agreement with the results of sheep rendered anosmic with ZnSO 4 (Hernandez, Serafin, Vazquez, Delgadillo, and Poindron, 2001). Despite the fact that the maintenance conditions differed (individual pens in the present work vs group rearing in the study of Hernandez et al., 2001), in both instances peripheral anosmia failed to affect global nursing parameters, its effects being limited to the absence of maternal selectivity. Altogether, these results are also in agreement with those reported for rats, in which the differential effects of peripherally induced anosmia and bulbectomy have been well documented (Alberts, 1974; Fleming and Rosenblatt, 1974; Wamboldt and Insel, 1987). On the other hand, our milk production results are contrary to those observed in cows: postpartum anosmia and privation of visual cues from the calf led to a decrease in milk production (Griffith and Williams, 1996). This difference may be due to the fact that, in this latter study, sensory deprivation occurred after the selective bond was already established, thus leading to more marked effects of anosmia, as already reported for maternal behavior in sheep (Poindron and Le Neindre, 1980; Alexander, Stevens, and Bradley, 1983). To conclude, the present results support the hypothesis that exteroceptive stimuli from the young play an important role in the modulation of maternal endocri-

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nology in the goat, as reported for other species, although the effects may vary between species and as a function of the lactational stage of the mother and the age of the young. In addition, the presence of a selective bond appears to be an important element in this modulation. Whether sensory stimuli from the young exert their controlling action on maternal motivation by long-term effects on maternal endocrinology remains an open question. Longitudinal studies investigating how the effects of suckling on lactation, first excitatory and then inhibitory, may affect neural structures involved in the control of maternal behavior may help to clarify this point.

ACKNOWLEDGMENTS The authors thank Dwigtht Krebhiel, Ph.D., for his support in the data analysis and Richard Porter for the final revision of the English version of the manuscript. We are also grateful to J. A. Flores and A. D. Rodrı´guez for their help in collecting data and to Rau´ l Paulin for taking care of the animals. INTERVET Mexico provided us with Chrono-gest (sponges containing fluorogestone acetate) and Folligon (PMSG hormone) to synchronize the reproduction of the animals. This study was supported by PAPIIT-UNAM, Grant IN212796, and by the Veterinary School of the Autonomous University of Queretaro. H. Hernandez was supported by a CONACyT scholarship for doctoral studies.

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