Maternal Olfaction Differentially Modulates

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

Oxytocin and Prolactin Releaseduring Suckling in Goats

Horacio Hernandez,* Norma Serafin,* Angelica M. Terrazas,*Pierre Guy Marnet,† Guy Kann,‡ Jose Alberto Delgadillo,§and Pascal Poindron*,1

*Centro de Neurobiologıa, Universidad Nacional Autonoma de Mexico, AP 1-1141,Juriquilla, Queretaro 76001 Qro., Mexico; †Unite mixte INRA/ENSAR de Recherches sur laProduction du Lait, INRA, 65, rue de Saint-Brieuc, 35042 Rennes Cedex, France; ‡Unite deRecherches sur l’Endocrinologie du Placenta et de la Perinatalite, Laboratoire de BiologieCellulaire et Moleculaire, INRA, 78352 Jouy en Josas Cedex, France; and §Departamentode Ciencias Medico Veterinarias, Universidad Autonoma Agraria Antonio Narro,Carretera a Santa Fe y Periferico, AP 940, Torreon Coahuila, Mexico

Received January 21, 2002; accepted March 11, 2002

In postparturient goats, olfactory recognition of theyoung allows the establishment of a selective bond be-

the OT, but not the PRL, response to suckling. In addi-tion, perception of the smell of the young appears to

tween the mother and her kids. Once this bond isformed, the mother rejects alien young that attempt tosuckle. We tested whether the development of the ma-ternal selective bond in goats modulates prolactin (PRL)and oxytocin (OT) release in response to suckling. Onday 37 of lactation, serial blood samples were takenduring nursing of the mother’s own or alien kid(s) in 10intact/selective goats and in 10 goats rendered anos-mic/nonselective through prepartum peripheral ZnSO4

irrigation. Spontaneous nursing behavior was also stud-ied weekly from day 7 to 30 of lactation, at which timemilk production was measured. Maternal selectivity hadno effect on PRL release, in contrast to OT release,which was significantly affected by this factor. Intactmothers released OT only when nursing their own kids,but not with aliens, while anosmic/nonselective damsshowed an increase in OT levels regardless of the iden-tity of the kids. In addition to these effects on maternalselectivity, the amplitude of the response of both hor-mones was lower in anosmic mothers than in intactmothers. Finally, nursing behavior and milk productionwere not significantly affected by anosmia. We concludethat maternal selective behavior in goats, which relies onthe individual olfactory signature of the kid, modulates

1 To whom correspondence and reprint requests should be ad-dressed at present address: UMR 6073, Physiologie de la Reproduc-tion et des Comportements, INRA, 37380 Nouzilly, France. Fax:52-4422-2381046. E-mail: poindron@servidor.unam.mx.

232

have a general facilitatory effect, independent of thekid’s identity, on the release of both hormones. © 2002

Elsevier Science (USA)

Key Words: goat; maternal behavior; suckling; prolac-tin; oxytocin; olfaction; anosmia; selectivity; lactation.

Mammals show different patterns of mother–younginteractions in relation to the developmental status ofthe neonate (Gubernick, 1981; Gonzalez-Mariscal andPoindron, 2002). Thus, in altricial species (most ro-dents and lagomorphs), the mother builds a nest inwhich she gives birth to offspring that are not welldeveloped. Mother–young interactions occur withinthe nest and in general do not depend strongly onmutual recognition. On the other hand, precocial spe-cies (e.g., ungulates) give birth to well-developed off-spring, which stand and walk as early as 30 min afterbirth and start to suckle within 1 h. Moreover, anexclusive and enduring bond between the mother andher offspring is rapidly established shortly after par-turition. Thereafter, the mother accepts only her ownyoung at the udder and actively rejects any alien thatattempts to suckle (this aspect of maternal behavior isreferred to as maternal selectivity; sheep, Bouissou,1968; goats, Hersher, Richmond, and Moore, 1963;cattle, Hudson and Mullord, 1977). It is also well es-

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tablished that in sheep and goats, this maternal selec-tivity depends on the individual olfactory signatureof the offspring (Poindron and Le Neindre, 1980;Porter, Levy, Poindron, Litterio, Schaal, and Beyer,1991; Porter, Romeyer, Levy, Krehbiel, and Nowak,1994; Romeyer, Porter, Levy, Nowak, Orgeur, andPoindron, 1993).

In mothers of altricial species, which do not developselective nursing, it is well established that the stimu-lus caused by suckling of the pups induces the releaseof prolactin (PRL) and oxytocin (OT) into the periph-eral blood (Grosvenor, Shyr, Goodman, and Mena,1986; Fuchs, Cubile, Dawood, and Stener-Jørgensen,1984). These hormones stimulate the synthesis andejection of milk from the mammary glands. Neverthe-less, in the lactating rat, PRL release is not exclusivelycaused by the suckling stimulus of the offspring, sinceperception of the pups’ olfactory cues alone can elicitPRL release from the maternal pituitary gland similarto that induced by suckling (Grosvenor, 1965; Menaand Grosvenor, 1971). Also, in certain circumstances,auditory cues from the pups may be important forPRL release (Terkel, Damasa, and Sawyer, 1979; Sternand Siegel, 1978; Stern, Thomas, Rabii, and Barfield,1984). Finally, toward the final phase of lactation, ithas also been reported that mother rats may releasePRL in response to exteroceptive stimulation, fromboth their own pups and alien pups of other lactatingmothers (Mena and Grosvenor, 1972).

While the effect of suckling on PRL and OT releaseis also well documented in species that establish anexclusive bond with their litter (goats: Buttle, Forsyth,and Knaggs, 1972; McNeilly, 1972, sheep: Kann,Habert, Meusnier, and Ryniewicz, 1977; cattle: Akersand Lefcourt, 1982), there is little information concern-ing the relationships between the presence of maternalselective behavior and the endocrinology of lactation.Indications that exteroceptive stimuli can induce hor-monal release in lactating females have also been re-ported in these species. In goats, Hart (1974) suggestedthat PRL release can be induced not only by the tactilestimulation of the teats due to suckling, but also due toa conditioned response associated with the eventswhich take place just before milking or in response toa metabolic stimulus. Nevertheless, in a later studyHart and Linzell (1977) showed that stimuli associatedwith the milking routine did not appear to induce aconditioned released of PRL. Also, mechanical stimu-lation of the teat by milking or suckling did not inducePRL or OT release in females in which the udder hadbeen denervated (Kann, 1980). In addition, McNeilly(1972) found that few goats (2 of 12) responded with a

conditioned release of oxytocin to exteroceptive cuesfrom kids. On the other hand, there are some indica-tions that maternal selectivity might influence PRL orOT release at the time of suckling. For instance, incattle, Perez, Jimenez de Perez, Poindron, Le Neindre,and Ravault (1985) showed that the PRL response tomachine milking was significantly lower than thatinduced by nursing of the mother’s own calf and thatsuckling by an alien calf did not lead to any significantresponse. Also, Silveira, Spoon, Ryan, and Williams(1993) found that the proportion of cows experiencingOT release following controlled suckling was greaterin mothers nursing their own calf than in dams forcedto nurse an alien calf.

Goats, like sheep and cattle, also develop a selectivematernal behavior within the first hours postpartumon the basis of olfactory cues from the kids, and moth-ers made anosmic before parturition do not developmaternal selectivity (Romeyer, Poindron, and Orgeur,1994; Hernandez, 2001). Nevertheless, whether mater-nal selectivity modulates PRL and OT release in re-sponse to suckling depending on the identity of theyoung has not been investigated thus far in this spe-cies. Also, while maternal olfaction appears to be ofmajor importance for the establishment of the mater-nal bond, it is not known whether prepartum anosmiamay result in detrimental effects on nursing behavior,as previously reported in sheep (Poindron, 1976a). Thepurposes of the present study were therefore (1) todetermine whether maternal selectivity modulates thePRL and OT response to suckling, depending on theidentity of the kid, in selective (intact) and nonselec-tive (anosmic) lactating goats, and (2) to assess anypossible effect of anosmia on nursing behavior.

METHODS

Animals

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

233Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

Anosmia Procedure

In anosmic goats, the olfactory mucosa was de-stroyed by two successive intranasal irrigations with asolution of zinc sulfate (1.5%) and procaine chlorhy-drate (3%) under general anesthesia (Romeyer et al.,1994) at a 1 week interval. The first treatment wasperformed 1 month before the expected date of par-turition. Olfactory deficits were confirmed 24 h post-partum and on day 30 of lactation with a 2-min be-havioral feed preference test (Poindron, 1976b). Eachfemale was tested individually, giving her the choicebetween two buckets, both containing concentrate pel-lets, but one with the upper part of its walls coatedwith dog excrement that was not in contact with thefood. All animals treated were found to be anosmicaccording to this test (i.e., consumed food associatedwith the odor of dog excrement for more then 10 s,while intact animals did not consume any) during thetwo postpartum periods.

Maintenance Conditions and PostpartumSelectivity Test

Animals were fed lucerne hay and concentrate (Ge-neraleche, with 18% crude protein, Purina), with min-eral supplements and water available ad libitum. Ra-tions were adjusted according to the goats’physiological status (stage of pregnancy and lacta-tion), as well as for litter size (NRC, 1981). Beforeparturition, animals were kept under intensive man-agement at a density of 5 m2/animal. Immediatelyafter parturition, mothers and their kid(s) were placedin contiguous individual pens (2 � 2 m) providedwith shade, where they remained until the end of thestudy.

To ensure that anosmia was associated with theabsence of selectivity, all mothers were tested for theirresponse to an alien kid at 24 h postpartum. To thisend, each of the mothers in both groups was separatedfrom her kid(s) and placed in a different testing pen(2 � 2 m) for 5 min. A kid (own or alien, in a balancedorder in each group) was then introduced and behav-ior of the female was observed for 2 min. The behav-iors registered were as follows: number of low- andhigh-pitched bleats, acceptance at the udder, durationof nursing, rejection at the udder, head butts, andthreats.

Nursing Behavior

The first monitoring of spontaneous nursing beganon day 7 after parturition and was followed by three

further periods of observation at intervals of 7 � 1days, each session lasting 2 h/mother. The onsetof observations varied from 08:00 to 10:00 h andwas randomized between groups. When observingmothers with twins, one observer followed only onemother at any one time. In the case of the motherswith single kids, two mothers were followed at thesame time. The observers were located in a passagecontiguous to the individual pens. In the present arti-cle, we use the terminology proposed by Hall, Hud-son, and Brake (1986), where the term nursing refers tothe activity of the mother and suckling to the activity ofthe young. Identity of the kid, duration of suckling foreach kid, and rejections were recorded on paper. Re-jection rate was calculated by dividing the number ofrejections by the sum of the number of successfulnursing episodes plus the number of rejections at theudder. The nursing parameters were defined in accor-dance with the criteria from Delgadillo, Poindron,Krehbiel, Duarte, and Rosales (1997). A nursing epi-sode was defined as the period during which a damnursed without interruption for more than 3 s, regard-less of the number of kids that suckled. Rejection wasdefined as any attempt by a kid to suckle that lastedless than 3 s after its beginning and was interrupted bythe dam either moving away or responding aggres-sively toward the kid. The behavioral parameters as-sessed for nursing (mothers) and suckling (kids) weretheir frequency, mean, and total duration/2 h, as wellas the rejection rate (%) for nursing attempts.

Hormonal Response to Suckling on Day 37of Lactation

The serial blood samples to study the PRL and OTresponses to suckling were obtained in the individualpen in which the mother was normally staying withher young. A catheter was inserted into the jugularvein of each female at 24 h before sampling and fixedin the vein of the animal by sewing it on the skinunder local anesthesia (2% Xylocaine). Heparin wasused as an anticoagulant to keep the catheter func-tional. At the time of sampling and during the time theyoung were present in the pen, the mothers weregently restrained, always by the same person to min-imize stress due to handling. Also, when the youngwere introduced in the pen and at the beginning ofsuckling, the mother’s head was positioned so that hernose was less than 2 cm away from the body of theyoung, and she was allowed to smell them during theentire time they were present in the pen. Thus, thesampling conditions were the same in all cases, re-

234 Hernandez et al.

gardless of the group and of the identity of the kids.All blood samples were obtained in the morning be-tween 08:00 and 11:00 h. On each occasion, 2 h beforethe sampling procedure, the kids (own or alien) to beused for suckling were removed from their motherand placed in a distant pen out of sight of the mother.However, acoustical isolation was not possible and thebleats emitted by the kids due to separation could beheard by the dam. Samplings with the own and thealien kids were made on 2 consecutive days and theorder of the type of kid was balanced in both groups.The first two samples were taken at 3 and 2 min beforethe onset of suckling, this latter time being consideredas time 0. After the second sample was obtained, thekids were held in front of the female for 2 min, duringwhich time she could see, hear, and smell the kid(s)but not nurse. A third sample was taken after the firstminute of kid exposure and a fourth at the end of thisperiod, 1 min later. The kids were then immediatelyreleased and allowed to suckle. Additional blood sam-ples 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 stimu-lation, which consisted of 2 min of suckling by theown kid or an alien kid of the same age. In the case ofmothers with twins, both kids were used for the udderstimulation and two alien kids for the alien condition.Five milliliters of blood was collected for each hor-mone in tubes kept on ice. After centrifugation at 4000rpm at 4°C, plasma was stored at �20°C until hor-monal assay.

Hormonal Assays

Plasma PRL concentrations were determined byRIA in accordance with the method described by Kann(1971). The limit of detection was 1.24 ng/ml�1, andthe inter- and intra-assay coefficients of variation were4.5 and 12.6% for 1.36 ng/ml�1, respectively. PlasmaOT levels were determined by enzyme immunoassay(Marnet, Volland, Pradelles, Grassi, and Beaufils,1994). The limit of detection was 1.5 pg/ml�1, and theinter- and intra-assay coefficients of variation were 13and 8.6% for 4 pg/ml�1, respectively.

Milk Production at 1 Month of Lactation

In all mothers, milk production was assessed at 1month of lactation, based upon differential bodyweight of the kids before and after suckling over aperiod of 24 h (Ricordeau, Boccard, and Denamur,1960). The day before initiating the measurements, afirst emptying of the udder was performed at 18:00 h,

followed by three controlled sucklings of 4 min eachon the following day at 08:00, 13:00, and 18:00 h.Moreover, after each controlled suckling, 2 IU of ex-ogenous OT was injected into the jugular vein, fol-lowed by hand milking to extract any residual milk.The weight of this residual milk was added to that ofthe corresponding body weight measurement.

Statistical Analysis

The behaviors recorded in the selectivity tests wereanalyzed using the Mann–Whitney test for indepen-dent data and the Wilcoxon test for two relatedsamples. Nursing and suckling variables from spon-taneous maternal behavior were transformed tologarithms to approximate a normal distributionand analyzed with a two-way repeated-measuresANOVA. Hormonal data were analyzed using two-way MANOVA (group as between-factor and time ofsampling and identity of the kids as within-factors) todetect general effects. Moreover, the effect of kid iden-tity within each group was analyzed by two trialfactor MANOVA. Likewise, the group effect was an-alyzed for each type of kid (own or alien) using atwo-way MANOVA (group as between-factor andtime of sampling as within-factor). For OT levels, thedata were first approximated to a normal distributionthrough logarithm transformation (Moore and Mc-Cabe, 1989). Milk production on day 30 of lactationwas compared between groups with a t test. All sta-tistical analyses were performed using the packageSystat 7.0 (SPSS, Chicago). Results are given asmeans � standard error (SEM).

Ethical Note

Animal care and experimental treatments compliedwith Guidelines A37801 of the French Ministry ofAgriculture for animal experimentation, which werealso used in a former collaborative study with Frenchscientists concerning the effects of anosmia on mother–young recognition in sheep in the frame of a col-laborative work (Ferreira, Terrazas, Poindron, Nowak,Orgeur, and Levy, 2000). In addition, anosmia neitheraltered the growth rate of the goats during the last 2months of pregnancy nor induced abortions or anyobservable changes in the general behavior of thefemales, which is consistent with what has been pre-viously reported in sheep (Hernandez, Serafin, Vasquez,Delgadillo, and Poindron, 2001). Furthermore, no dif-ferences were detected between groups in the birthweight of the kids. None of the selectivity tests re-

235Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

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

RESULTS

Maternal Behavior Selectivity Test at 24 hPostpartum

In intact mothers, all behaviors except maternalbleats differed significantly between the own andalien kid conditions (Wilcoxon test, P � 0.05 in allcases). On the contrary, this was not the case in anos-mic mothers. Furthermore, the behaviors of the moth-ers toward their own kid did not differ significantlybetween intact and anosmic dams, whereas differ-ences were observed in their behavior toward the alienkids (Mann–Whitney test, P � 0.05; Table 1). Alto-gether, these results show that anosmia was actuallyassociated with the absence of selective behavior inthis group.

Spontaneous Behavior of the Mothers andTheir Kids

Results of the observations of spontaneous maternalnursing behavior and suckling behavior by kids arepresented in Table 2. No significant differences werefound in the nursing behavior parameters betweenintact and anosmic goats at any time during the study(P � 0.87, for all variables; Table 2). On the otherhand, a significant effect of time (but no interactionbetween time and group) was found regarding thefrequency of nursing episodes, which decreased in

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

Hormonal Response to Suckling

A preliminary comparison (MANOVA) of hor-monal levels obtained in multiparous and primipa-rous goats within each group did not reveal any effectof the previous number of parturitions, neither forPRL nor OT (P � 0.20). Similarly, no effect of thenumber of kids raised by the dam was found for eitherhormone (P � 0.19). Finally, the presence of theyoung during the 2 min preceding suckling had noeffect on prenursing levels of PRL or OT. Therefore,the basal levels of these hormones were defined as themean of the three (PRL) or four (OT) prestimulationvalues.

PRL. Results for this hormone are presented inFig. 1. In intact mothers, the PRL concentrations in-creased from basal levels of 58 � 13 ng/ml and 72 �12 ng/ml to maximum values of 112 � 18 ng/ml and116 � 9 ng/ml for own and alien kids, respectively. Inanosmic dams, the corresponding values were 77 � 12ng/ml and 66 � 11 ng/ml for basal levels and 93 � 11ng/ml and 85 � 11 ng/ml for maximum levels. Thegeneral analysis revealed a main effect of the time ofsampling on PRL concentration (P � 0.001; Fig. 1),but no effect of group or identity of the kid. On theother hand, a significant interaction was found be-tween group and time of sampling (P � 0.014; Fig. 1),indicating some effect of anosmia on the amplitude ofrelease of this hormone. In intact dams, PRL levelswere increased both during suckling by the own kids

TABLE 1

Behavioral Responses (Mean � SEM) of Intact and Anosmic Goats Toward Own and Alien Kid in a 2-min Test of Maternal Acceptanceat the Udder Performed at 24 h Postpartum

Behavior

Intact goats (n � 10) Anosmic goats (n � 10)

Own P Alien Own P Alien

Udder acceptance (frequency) 1.8 � 0.4 * 0.1 � 0.1a 1.5 � 0.3 n.s. 0.7 � 0.2b

Nursing time (s) 29.0 � 11.7 * 0.7 � 0.7 29.0 � 10.3 n.s. 15.0 � 10.3Low-pitch bleats (frequency) 6.7 � 1.7 n.s. 6.3 � 2.6a 7.2 � 2.1 * 11.3 � 1.8b

High-pitch bleats (frequency) 2.8 � 2.2 * 14.0 � 5.4a 0.2 � .02 n.s. 2.3 � 2.1b

Aggressive behavior (frequency) 0 * 2.3 � 1.3a 0 n.s. 0.4 � 0.4b

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).

236 Hernandez et al.

and during suckling by the alien kids, whereas inanosmic mothers, suckling by the kid(s) induced asmaller response, which was significant only in thecase of the alien kid(s).

OT. Results for this hormone are presented in Fig.2. In the intact group, OT basal levels were 33 � 14pg/ml and 16 � 3 pg/ml for the own and the alienkids, respectively. In the anosmic group, basal levelswere 17 � 2 pg/ml and 22 � 8 pg/ml, and peak levelswere 49 � 7 pg/ml and 49 � 12 pg/ml, for own andalien kids, respectively. While the prestimulationbasal levels of this hormone in the selective groupappeared to be higher with the own kid than with thealien, this was not significant (P � 0.14, Wilcoxon)and it is important to stress that this difference wasdue to one single goat, which presented a high OTlevel (143 pg/ml) before the introduction of her ownkid. Furthermore, excluding this animal did notchange the significance of any of the analyses.

The MANOVA revealed significant main effects ofidentity of the kid (P � 0.02) and time (P � 0.001),as well as a significant interaction between time ofsampling and identity of the kid (P � 0.001) and atendency for an interaction between group, time, and

identity of the kid (P � 0.06). Indeed, a significanteffect of anosmia on the amplitude of release wasfound in the case of suckling by the own kid: the OTresponse was different depending on the identity ofthe kids in intact/selective mothers, while in anos-mic/nonselective dams this was not the case (Fig. 2).In intact mothers the response induced by the ownkids consisted of an increase in OT basal levels 1 minafter the onset of nursing. By contrast, these intactdams showed no significant changes in OT levels inresponse to suckling by alien kids. In anosmic moth-ers, on the other hand, a main effect of identity of thekids was not found (P � 0.1), whereas there was aclear 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 periodby the mothers did not differ between intact and anos-mic goats (1.7 � 0.2 kg and 1.9 � 0.3 kg, respectively;ANOVA, P � 0.99). On the other hand, a significantdifference was found between mothers of a single kidand mothers of twins (1.29 � 0.20 kg and 2.01 � 0.20

TABLE 2

Characteristics of Spontaneous Nursing (Mothers) and Suckling (Kids) Behaviors (mean � SEM) in Intact and Anosmic Crossbred DairyGoats during the First Month of Lactation

Time postpartumEvolutionwith timeWeek 1 Week 2 Week 3 Week 4

MothersIntact group

Number of nursing episodes/dam/2 h 8.5 � 2.7 4.7 � 0.8 2.8 � 0.5 3.2 � 0.6 P � 0.05Total duration of nursing episodes/dam (min) 3.3 � 0.9 2.1 � 0.7 1.4 � 0.3 2.2 � 0.9 n.s.Mean duration of a nursing episode/dam (s) 25.7 � 3.5 24.2 � 5.4 30.1 � 5.6 41.0 � 16.7 n.s.Rejection rate at nursing (%) 2.3 � 1.2 28.9 � 10.2 42.6 � 10.5 43.4 � 10.6 P � 0.05

Anosmic groupNumber of nursing episodes/dam/2 h 8.1 � 2.2 4.0 � 0.8 3.5 � 0.5 4.0 � 1.0 P � 0.05Total duration of nursing episodes/dam (min) 4.8 � 1.6 2.2 � 0.5 1.5 � 0.2 1.5 � 0.3 P � 0.05Mean duration of a nursing episode/dam (s) 32.1 � 5.7 28.6 � 4.3 26.2 � 1.7 25.0 � 4.3 n.s.Rejection rate at nursing (%) 2.9 � 2.1 37.5 � 7.9 31.1 � 8.8 31.0 � 7.3 P � 0.05

KidsIntact group

Total number of suckling episodes/kid/2 h 5.4 � 1.5 3.5 � 0.7 2.7 � 0.5 3.3 � 0.4 n.s.Total duration of suckling/kid (min) 2.1 � 0.5 1.6 � 0.4 1.2 � 0.2 2.0 � 0.5 n.s.Mean duration of a suckling period/kid (s) 24.4 � 3.1 22.0 � 10.5 25.7 � 3.5 39.0 � 10.6 n.s.

Anosmic groupTotal number of suckling episodes/kid/2 h 6.1 � 1.4 3.4 � 0.5 3.6 � 0.4 4.1 � 0.7 n.s.Total duration of suckling/kid (min) 3.4 � 0.9 1.7 � 0.3 1.5 � 0.2 1.6 � 0.2 n.s.Mean duration of a suckling period/kid (s) 28.3 � 4.8 29.3 � 2.9 25.6 � 1.8 26.0 � 3.2 n.s.

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

237Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

kg, P � 0.05), but no interaction between group andnumber of kids.

DISCUSSION

Two main conclusions can be drawn from our re-sults. First, the study carried out in intact mother goatsindicates that maternal selectivity does not exert thesame control on PRL and OT release induced by suck-ling. While the identity of the young that suckles hasno influence on prolactin release, it is of major impor-tance for the release of oxytocin. This is further con-firmed by the fact that prepartum anosmia in themother results in a similar and significant increase of

OT levels when the mother is suckled by her own kidor by an alien kid, whereas the release of prolactinremains similar for the two types of kids. The secondpoint revealed by our study is that the absence ofolfactory perception in the mother is associated withglobally lower levels of both PRL and OT in responseto suckling of the own young, while it has no effect onmilk production or global nursing activity. As awhole, it appears therefore that the exteroceptive ol-factory stimuli from the kids have a differential anddual modulatory action on the secretion of these hor-mones in response to suckling: on the one hand, theperception of these olfactory cues has an overall facili-tatory action on the release of both hormones whenthe mother is nursing her own young, and, on the

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

238 Hernandez et al.

other hand, the perception of the individual smell ofan alien kid totally blocks the release of OT. To ourknowledge, this is the first time that such an interac-tion between exteroceptive and proprioceptive stimulion PRL and OT secretion has been reported in goatsand that the effects of maternal anosmia on theseparameters have been investigated in this species. Ourpresent results are also in agreement with the obser-vations made previously in dairy Lacaune ewes sub-mitted to a mixed system of suckling and milking(Marnet and Negrao, 2000). These authors found thatmilking in maternal ewes was not followed by OT

release, while cortisol release and prolactin releasewere maintained. Our results are also congruent withthe absence of conditioned OT release to milkingfound in this species by Marnet, Negrao, and Labus-siere (1998).

Effects of Maternal Selectivity on HormonalRelease

Regarding OT, our results are consistent with theidea proposed for other species that the release of thishormone can be affected by the expression of maternal

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

239Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

exclusive acceptance of the young at the udder. Thus,in cattle, which also show selective nursing (Hudsonand Mullord, 1977; Le Neindre and Garel, 1979), theproportion of dams showing OT release followingsuckling by the own calf on day 4 of lactation washigher than that of dams forced to nurse an alienyoung (Silveira et al., 1993). These results are opposedto those found in the rat, in which lactating motherswill nurse alien pups even though they are able todiscriminate between them and their own offspring(Beach and Jaynes, 1956). For example, Grosvenor et al.(1986) found a significant increase in the peak plasmaOT concentration following suckling by a litter of sixhungry alien pups, even after the mother had nursedher own pups. This is further supported by the factthat in the present study, prepartum anosmia, whichimpedes the establishment of selective nursing ingoats (Klopfer and Gamble, 1966; Romeyer et al.,1994), resulted in the release of OT in response tosuckling by an alien kid, even though anosmic moth-ers are still able to discriminate their own young onthe basis of nonolfactory cues (Gilling, 2002). OT re-lease in anosmic mothers also indicates that the failureto observe OT release in the presence of an alien kid inintact mothers is not due to the absence of the indi-vidual odor of the own kid. If this were the case, OTresponse by anosmic dams would not have been ob-served in the presence of either type of kid.

Taken together, our results indicate therefore thatindividual olfactory cues from the young, which arethe basis for maternal selectivity, also modulate therelease of OT at the time of nursing. It is important tostress, however, that the presence of exteroceptivecues by themselves did not promote the release of OTor PRL, contrary to what has been previously reportedfor PRL in the rat (Mena and Grosvenor, 1971; Terkelet al., 1979) and also in mastectomized cattle (Steven-son, Knoppel, Minton, Salfen, and Garverick, 1994). Inaddition, the modulation of suckling-induced releaseof PRL by maternal selectivity does not appear asconsistent as for OT. In the present study, we failed tofind any effect of the identity of the kid on this vari-able, which is consistent with results reported by Sil-veira et al. (1993) in cattle, but not with those of Perezet al. (1985), who found that suckling by an alien calfdid not lead to any significant PRL response.

General Effects of Anosmia on Hormonal Release

The results obtained in anosmic mother goats sug-gest that the influence of olfactory cues from theyoung is not limited to their participation in maternal

selectivity. While no positive effects of exteroceptivestimuli were observed in the absence of suckling,anosmia resulted in a significantly smaller release ofPRL in response to the suckling by the own and thealien young and also in a lower OT response to suck-ling by the own young. It appears therefore that inaddition to the selectivity-dependent effect of olfac-tory cues on OT release, olfactory perception has ageneral positive effect on PRL and OT release inducedby suckling. Such an effect of anosmia is similar to thatreported in the rat for PRL, although in this latterspecies the presence of olfactory cues from the pupswas sufficient to induce hormonal release in intactdams (Mena and Grosvenor, 1971). In addition, de-spite the fact that in the present study exteroceptivestimuli from the young were unable to induce a re-lease of OT independently of suckling, this does notexclude the possibility that such a release may occur.Thus, McNeilly (1972) found that some goats (2 of 12goats in his study) showed an OT release before suck-ling, due to exteroceptive sensory stimulation fromthe kids. Similarly, in sheep, OT release was inducedby exteroceptive stimulation emanating from the lamb(Fuchs, Ayromlooi, and Rasmussen, 1987). One possi-ble reason for the differences between these resultsand those of the present work may be the day oflactation on which the studies were performed. Thus,in the study by Fuchs et al. (1987) the samples wereobtained between days 1 and 15 postpartum versusbetween day 1 and 21 of lactation in the study ofMcNeilly (1972) and on day 37 postpartum in ourstudy. In species such as rats and rabbits, it seems thatthe ability of either suckling or exteroceptive stimula-tion from the pups to modulate PRL release or lacta-tion duration depends on the lactation stage of themother and the age of the pups (Grosvenor, Shah, andCrowley, 1991; Mena, Clapp, and Martınez de la Es-calera, 1990; Mena, Clapp, Aguayo, Morales, and Mar-tınez de la Escalera, 1991).

An alternative to the hypothesis of a direct involve-ment of olfaction in the modulation of PRL and OTrelease is that behavioral stress affects the response ofthese hormones to suckling in intact goats. Suckling byan alien kid may be quite a stressful event for an intactdam, thus resulting in the release of �-endorphin andinhibition of OT release, as well as in stress-inducedPRL release. However, although the inhibitory influ-ences of opiates on OT release are well established inthe rat (Wakerley, 1994; Russell, Coombes, Leng, andBicknell, 1993), their exact role in goats or ruminantsin general is not clearly understood (Aurich, Dobrin-ski, Hoppen, and Grunert, 1993; Bruckmaier and

240 Hernandez et al.

Blum, 1996; Bruckmaier, Shams, and Blum, 1993; Gor-don, Renfree, Short, and Clarke, 1987; Hashizume,Haglof, and Malven, 1994; Seckl and Lightman, 1987).Also, if stress was responsible for the PRL responseobserved in intact mothers nursing an alien kid, dif-ferential effects of anosmia should have been foundbetween the responses to own vs alien young. There-fore, if some effect of stress cannot be excluded, ourresults appear more in concordance with a specificmodulation of hormonal release by olfactory stimuli.

The neurobiological mechanisms that control thedifferential effects of olfaction on the suckling-inducedrelease of PRL and OT in goats are not known. Severalstudies in rats have provided evidence for a directsynaptic input from the main and accessory olfactorysystems to supraoptic nuclei, which could be involvedin the modulation of OT release by olfactory cues(Hatton and Yang, 1989; Smithson, Weiss, and Hatton,1989; Smithson, Weiss, and Hatton, 1992). Also, it isinteresting to note that some parallels can be madebetween our results and those concerning the electro-physiological responses found in the olfactory bulb ofthe postparturient sheep, a species in which selectivityof maternal behavior also depends on olfaction. Whilea large proportion (70%) of cells respond to the odor ofany lamb, others (30%) respond preferentially to theodor of the ewe’s own lamb (Kendrick, Levy, andKeverne, 1992). Moreover, when a dam established aselective bond with her lamb, its odor, but not that ofaliens, produced an increase in the release of bothglutamate and GABA in the olfactory bulbs. By con-trast, both own and alien lamb odors produce anincrease of acetylcholine and noradrenaline release,suggesting that these two latter neurotransmitters areimportant for general lamb odor identification, whileglutamate and GABA are involved in the selectiveolfactory recognition of the lamb. Therefore, it is pos-sible that the endocrine effects found in goats maydepend on a similar differential activation of the firstolfactory relay located in the olfactory bulbs. If thishypothesis is correct, it would also be congruent withthe absence of differences in the hormonal responsesof primiparous and multiparous mothers, since at theneurobiological level, the differences reported in theolfactory bulb disappear within a few hours postpar-tum (Levy, Porter, Kendrick, Keverne, and Romeyer,1996). Our results are also in agreement with those ofNegrao, Marnet, and Kann (1998), who reported thatthe PRL response to milking by dairy sheep did notdiffer between the first and the second lactation in thesame animals.

Effects of Anosmia on Behavior and MilkProduction

In the light of the differences found between intactand anosmic mothers in their PRL and OT responsesto suckling, it may seem surprising that milk produc-tion did not differ between the two groups. However,since neither PRL or OT responses were totally sup-pressed, it is likely that sufficient levels of both hor-mones were released for initial lactogenesis, as well asfor maintenance of milk synthesis and udder evacua-tion. This is further supported by the fact that udderevacuation, an essential process for maintenance ofmilk production (Wilde and Peaker, 1990; Peaker andWilde, 1996), does not appear to depend as stronglyon OT release in the goat as it does in other speciesthat have a smaller cisternal milk fraction (Marnet andMcKusick, 2001). It is also consistent with the fact thatanosmia did not significantly modify maternal nurs-ing activity, contrary to the results reported earlier forsheep (Poindron, 1976a), in which nursing activitywas decreased in anosmic mothers. A likely reason forthis difference is the fact that anosmia was inducedperipherally by irrigation of the olfactory mucosa withZnSO4 versus olfactory bulbectomy in the study ofPoindron (1976a). This is also in agreement with theresults of sheep rendered anosmic with ZnSO4 (Her-nandez, Serafin, Vazquez, Delgadillo, and Poindron,2001). Despite the fact that the maintenance conditionsdiffered (individual pens in the present work vs grouprearing in the study of Hernandez et al., 2001), in bothinstances peripheral anosmia failed to affect globalnursing parameters, its effects being limited to theabsence of maternal selectivity. Altogether, these re-sults are also in agreement with those reported forrats, in which the differential effects of peripherallyinduced anosmia and bulbectomy have been well doc-umented (Alberts, 1974; Fleming and Rosenblatt, 1974;Wamboldt and Insel, 1987). On the other hand, ourmilk production results are contrary to those observedin cows: postpartum anosmia and privation of visualcues from the calf led to a decrease in milk production(Griffith and Williams, 1996). This difference may bedue to the fact that, in this latter study, sensory depri-vation occurred after the selective bond was alreadyestablished, thus leading to more marked effects ofanosmia, as already reported for maternal behavior insheep (Poindron and Le Neindre, 1980; Alexander,Stevens, and Bradley, 1983).

To conclude, the present results support the hypoth-esis that exteroceptive stimuli from the young play animportant role in the modulation of maternal endocri-

241Maternal Olfaction Affects OT and PRL Release during Suckling in Goats

nology in the goat, as reported for other species, al-though the effects may vary between species and as afunction of the lactational stage of the mother and theage of the young. In addition, the presence of a selec-tive bond appears to be an important element in thismodulation. Whether sensory stimuli from the youngexert their controlling action on maternal motivationby long-term effects on maternal endocrinology re-mains an open question. Longitudinal studies investi-gating how the effects of suckling on lactation, firstexcitatory and then inhibitory, may affect neural struc-tures involved in the control of maternal behavior mayhelp to clarify this point.

ACKNOWLEDGMENTS

The authors thank Dwigtht Krebhiel, Ph.D., for his support in thedata analysis and Richard Porter for the final revision of the Englishversion of the manuscript. We are also grateful to J. A. Flores andA. D. Rodrıguez for their help in collecting data and to Raul Paulinfor taking care of the animals. INTERVET Mexico provided us withChrono-gest (sponges containing fluorogestone acetate) and Folli-gon (PMSG hormone) to synchronize the reproduction of the ani-mals. This study was supported by PAPIIT-UNAM, GrantIN212796, and by the Veterinary School of the Autonomous Uni-versity of Queretaro. H. Hernandez was supported by a CONACyTscholarship for doctoral studies.

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