Effects of intrauterine position during gestation on specific endocrine and behavioral parameters that impact reproduction in domestic rabbits.

Prior studies from others, performed in a different breed, reported that doe rabbits developing between two male siblings (2 M) during gestation display characteristics indicative of masculinization: larger anogenital distance (AGD), larger submandibular glands, and higher chinning frequency than females with zero (0 M) or one (1 M) contiguous brothers. Similar effects are provoked by injecting androgens to the pregnant doe suggesting that prenatal androgen exposure masculinizes female embryos. To further understand the scope of such masculinization we compared 0 M, 1 M, and 2 M females regarding behavioral, neuroendocrine, and somatic parameters, related or not to reproduction. IUP did not impact: body weight, sexual receptivity, mating-induced LH secretion, maternal nest-building, litter size, or milk output. At puberty: a) chinning frequency was: 0 M and males>1 M and 2 M; b) ambulation in open field was lowest in 1 M females and males. IUP effects on AGD were significant only on postnatal day 1: 0 M, 1 M, and males>2 M, in contrast to earlier study. Willingness to nurse at delivery was less frequent in 2 M than in 1 M and 0 M does and correlated with nursing occurrence across lactation. Does that did not nurse at parturition delivered fewer kits/min than those that nursed then, regardless of IUP. The duration of nursing bouts across lactation was significantly longer in the1 M and 2 M does that showed this behavior on postpartum days 1-20. Our findings indicate that IUP is associated with alterations in specific aspects of postpartum maternal behavior.

Oxytocin antagonist does not disrupt rabbit maternal behavior despite binding to brain oxytocin receptors.

We explored a possible role of oxytocin (OXT) for the onset and maintenance of rabbit maternal behavior by: (a) confirming that a selective oxytocin receptor antagonist (OTA) widely used in rodents selectively binds to OXT receptors (OXTR) in the rabbit brain and (b) determining the effect of daily intracerebroventricular (icv) injections of OTA to primiparous and multiparous does from gestation day 29 to lactation day 3. OTA efficiently displaced the high affinity, selective oxytocin receptor (OXTR) radioligand, 125 I-labeled ornithine vasotocin analog (125 I-OVTA), but was much less effective at displacing the selective V1a vasopressin receptor radioligand, 125 I-labeled linear vasopressin, thus showing high affinity and selectivity of OTA for rabbit OXTR as in rodents. Further, ICV OTA injections did not modify nest-building, latency to enter the nest box, time spent nursing or the amount of milk produced, relative to vehicle-injected does. The percentage of mothers suckling the litter was also similar between both groups, regardless of parity. Together, our results do not support a role of OXT for the initiation or maintenance of rabbit maternal behavior. Future studies are warranted to determine if OXT participates in fine-tuning additional aspects of the maternal ethogram, for example, circadian periodicity of nursing and nest defense.

Rabbit Maternal Behavior: A Perspective from Behavioral Neuroendocrinology, Animal Production, and Psychobiology.

Rabbit maternal behavior (MB) impacts meat and fur production on the farm, survival of the species in the wild, and pet welfare. Specific characteristics of rabbit MB (i.e., three-step nest building process; single, brief, daily nursing bout) have been used as models for exploring particular themes in neuroscience, like obsessive-compulsive actions, circadian rhythms, and cognition. Particular hormonal combinations regulate nest building by acting on brain regions controlling MB in other mammals. Nonhormonal factors like type of lodging and the doe's social rank influence nursing and milk production. The concurrency of pregnancy and lactation, the display of nonselective nursing, and the rapid growth of altricial young - despite a minimal effort of maternal care - have prompted the study of mother-young affiliation, neurodevelopment, and weaning. Neurohormonal mechanisms, common to other mammals, plus additional strategies (perhaps unique to rabbits) allow the efficient, adaptive display of MB in multiple settings.

Regulation of maternal behavior, social isolation responses, and postpartum estrus by steroid hormones and vaginocervical stimulation in sheep.

Periparturient ewes display several activities in addition to maternal behavior per se. They isolate themselves from the flock and, contrary to other mammals, do not show postpartum estrus. We investigated the possibility of a common hormonal control of maternal behavior, prepartum social isolation responses, and absence of postpartum estrus. We quantified responses to flockmate separation and incidence of sexual receptivity at several reproductive stages in intact ewes (Experiment 1). Responses to social isolation were lowest in preparturient ewes and at pregnancy day 149, intermediate at pregnancy day 147 and highest at day 136 and in non-pregnant ewes (P < 0.05 between the 3 levels). In a second experiment, we quantified the same parameters and maternal behavior in 1) ovariectomized ewes receiving medroxyprogesterone acetate only (ovxMPA); 2) ovariectomized ewes receiving MPA + estradiol benzoate (ovxSHORT); 3) intact ewes receiving a longer MPA + estradiol dipropionate treatment, before and after vaginocervical stimulation (VCS). Before VCS no steroid treatments decreased social isolation responses and maternal behavior was scarce or absent. Following VCS and interaction with lamb, maternal responses in the ovxSHORT group increased while social isolation responses decreased. Sexual receptivity occurred in non-pregnant ewes and in ovxSHORT group. Conclusion: some hormonal treatments +VCS can effectively induce maternal behavior and reduce social isolation responses. Long-term progestin treatment can inhibit postpartum estrus.

Behavioral, neuroendocrine and physiological indicators of the circadian biology of male and female rabbits.

Adult rabbits show robust circadian rhythms of: nursing, food and water intake, hard faeces excretion, locomotion, body temperature, blood and intraocular pressure, corticosteroid secretion, and sleep. Control of several circadian rhythms involves a light-entrained circadian clock and a food-entrained oscillator. Nursing periodicity, however, relies on a suckling stimulation threshold. Brain structures regulating this activity include the paraventricular nucleus and preoptic area, as determined by lesions and quantification of cFOS- and PER1 clock gene-immunoreactive proteins. Melatonin synthesis in the rabbit pineal gland shows a diurnal rhythm, with highest values at night and lowest ones during the day. In kits the main zeitgeber is milk intake, which synchronizes locomotor activity, body temperature, and corticosterone secretion. Brain regions involved in these effects include the median preoptic nucleus and several olfactory structures. As models for particular human illnesses rabbits have been valuable for studying glaucoma and cardiovascular disease. Circadian variations in intraocular pressure (main risk factor for glaucoma) have been found, with highest values at night, which depend on sympathetic innervation. Rabbits fed a high fat diet develop cholesterol plaques and high blood pressure, as do humans, and such increased fat intake directly modulates cardiovascular homeostasis and circadian patterns, independently of white adipose tissue accumulation. Rabbits have also been useful to investigate the characteristics of sleep across the day and its modulation by infections, cytokines and other endogenous humoral factors. Rabbit circadian biology warrants deeper investigation of the role of the suprachiasmatic nucleus in regulating most behavioral and physiological rhythms described above.

Maternal responsiveness to suckling is modulated by time post-nursing: A behavioural and c-Fos/oxytocin immunocytochemistry study in rabbits.

Doe rabbits nurse once/day for approximately 3 minutes, with circadian periodicity, inside the nest box. The amount of suckling received at each bout regulates this behaviour because reducing the litter size to four kits or less disrupts nursing circadian periodicity and increases suckling bout duration. Additionally, the likelihood that does will nurse kits at a given time of day depends on the time elapsed since the last suckling episode and the litter size nursed then. We hypothesised that the time elapsed since the last nursing would impact the number of c-Fos immunoreactive (IR) cells observed after suckling five kits and also that observed before the next nursing ("no kits"). Suckling significantly increased, relative to "no kits", the number of c-Fos-IR cells in the medial preoptic area and lateral septum but not in the bed nucleus of the stria terminalis (BNST), suprachiasmatic nucleus or ventromedial hypothalamus in does nursing at 18 or 24 hours after the previous bout. No effects of suckling were observed in mothers nursing at 6 hours. Does given kits at 3 hours post last suckling refused to nurse but, in the remaining three groups, all does nursed normally. At "no kits", more c-Fos-IR cells were seen (in all regions except the BNST) in does given kits at 24 hours after the last nursing and killed 1 hour later (ie, 4 hours after lights on) than in those killed earlier. The percentage of oxytocinergic (OT) cells co-expressing c-Fos was not modified by nursing in the paraventricular or supraoptic nuclei but, in the latter, the largest number of total OT-IR cells occurred at 18 and 24 hours post-last nursing. In conclusion, the responsiveness of particular forebrain regions involved in regulating circadian rhythms, lactation, and maternal behaviour is modulated by suckling and time of day.

Long-lasting behavioral and neuroendocrine changes provoked in rabbits by cancelling a single nursing bout in early lactation.

To restore estrus in lactating doe rabbits, nursing was prevented on lactation day 10 and allowed again from day 12 onward. This "biostimulation" procedure, used to accelerate reproduction, allows kit survival despite a 48 hr fast. Yet, the consequences of "biostimulation" on their psychobiological and neuroendocrine development are largely unknown. Therefore, we determined, in adult males and females, tested in the morning or afternoon: corticosteroid secretion (baseline and reactive), sexual behavior, and responses in six tests measuring stress/anxiety. The latter were not affected by maternal deprivation or time of testing in either sex. The lordosis quotient was reduced in deprived does (relative to controls), an effect eliminated when only non-kins (of both groups) were compared. Deprived males showed altered sexual behavior, evidenced by a large number of mounts not culminating in ejaculation. Corticosterone and cortisol secretion increased (relative to baseline) following i.m. saline in all groups, but the magnitude of the response was affected by maternal deprivation, time of day, and sex. Results indicate that particular behaviors and reactivity to stress have different thresholds regarding the effects of mother-litter separation in early lactation.

Circadian Rhythms and Clock Genes in Reproduction: Insights From Behavior and the Female Rabbit's Brain.

Clock gene oscillations are necessary for a successful pregnancy and parturition, but little is known about their function during lactation, a period demanding from the mother multiple physiological and behavioral adaptations to fulfill the requirements of the offspring. First, we will focus on circadian rhythms and clock genes in reproductive tissues mainly in rodents. Disruption of circadian rhythms or proper rhythmic oscillations of clock genes provoke reproductive problems, as found in clock gene knockout mice. Then, we will focus mainly on the rabbit doe as this mammal nurses the young just once a day with circadian periodicity. This daily event synchronizes the behavior and the activity of specific brain regions critical for reproductive neuroendocrinology and maternal behavior, like the preoptic area. This region shows strong rhythms of the PER1 protein (product of the Per1 clock gene) associated with circadian nursing. Additionally, neuroendocrine cells related to milk production and ejections are also synchronized to daily nursing. A threshold of suckling is necessary to entrain once a day nursing; this process is independent of milk output as even virgin does (behaving maternally following anosmia) can display circadian nursing behavior. A timing motivational mechanism may regulate such behavior as mesolimbic dopaminergic cells are entrained by daily nursing. Finally, we will explore about the clinical importance of circadian rhythms. Indeed, women in chronic shift-work schedules show problems in their menstrual cycles and pregnancies and also have a high risk of preterm delivery, making this an important field of translational research.

Bidirectional Effects of Mother-Young Contact on the Maternal and Neonatal Brains.

Adaptive plasticity occurs intensely during the early postnatal period through processes like proliferation, migration, differentiation, synaptogenesis, myelination and apoptosis. Exposure to particular stimuli during this critical period has long-lasting effects on cognition, stress reactivity and behavior. Maternal care is the main source of social, sensory and chemical stimulation to the young and is, therefore, critical to "fine-tune" the offspring's neural development. Mothers providing a low quantity or quality of stimulation produce offspring that will exhibit reduced cognitive performance, impaired social affiliation and increased agonistic behaviors. Transgenerational transmission of such traits occurs epigenetically, i.e., through mechanisms like DNA methylation and post-translational modification of nucleosomal histones, processes that silence or increase gene expression without affecting the DNA sequence. Reciprocally, providing maternal care profoundly affects the behavior, learning, memory and fine neuroanatomy of the adult female. Such effects are in many cases permanent and sometimes they involve the hormones of pregnancy and lactation. The above evidence supports the idea that the mother-young dyad exerts profound and permanent effects on the brains of both adult and developing organisms, respectively. Effects on the latter can be explained by the neural developmental processes taking place during the early postnatal period. In contrast, little is known about the mechanisms mediating the plasticity of the adult maternal brain. The bidirectional effects that mother and young exert on each other's brains exemplify a remarkable plasticity of this organ for organizing itself and provide an immense source of variability for adaptation and evolution in mammals.

Bilateral lesions of the paraventricular hypothalamic nucleus disrupt nursing behavior in rabbits.

Doe rabbits nurse the litter only once a day, for around 3 min, with circa 24-h periodicity. To explore the participation of the paraventricular nucleus (PVN) in regulating this behavior, we lesioned it bilaterally with kainic acid on lactation day 7. Bilateral lesions, comprising less than 50% of the total PVN volume, abolished nursing behavior (4/8 does) or severely disrupted its normal periodicity (4/8 does). In the latter case, duration of nursing bouts was normal. Body weight, food and water intake were not significantly affected by bilateral PVN lesions. Unilateral lesions of the PVN or lesions located outside this nucleus did not significantly alter nursing periodicity or any other behavioral parameter. Results indicate an important role of the PVN for (i) maintaining maternal behavior, and (ii) the periodic display of nursing across lactation in rabbits.

Estrogen-dependent estrous behavior in rabbits is antagonized by the antiprogestin RU486.

Estrogen receptor-alpha (ERα) dimerizes with unliganded progesterone receptor (PR) in target tissues to trigger genomic and non-genomic effects. In ovariectomized rats the antiprogestin RU486 or antisense nucleotides against PR antagonize estradiol-induced sexual receptivity. We determined the relevance of unliganded PR for the expression of estrogen-dependent scent-marking (chinning) and sexual receptivity by injecting RU486 to: a) ovariectomized (ovx) rabbits given estradiol benzoate (EB; 5µg/day); b) intact rabbits. Chinning and lordosis were quantified in ovx animals before (5days; baseline) and during hormonal treatments: EB+RU486 (20mg/day; n=18) or EB+vehicle (n=18). On treatment day 4 LQ (lordosis quotient) increased in both groups, relative to baseline (mean±se): LQ=1±5 (baseline) vs 25±21 (EB+RU486) and 2±6 (baseline) vs 37±29 (EB+vehicle). On day 9 LQ values were: 22±23 (EB+RU486) and 54±39 (EB+vehicle). Chinning increased only in the EB+vehicle group (day 9=55±46 vs baseline=17±20 marks/10min). In intact rabbits one RU486 injection: reduced the LQ from 72±7to 36±8 five hrs later, increased the latency to receive first ejaculation from 45 to 98s, and decreased the number of ejaculations received in the test from 3 to 2 but did not modify mounting latency or chinning. Results support a participation of unliganded PR for the induction (ovx) and maintenance (intact) of rabbit estrous behavior by estrogens.

Neuroanatomical distribution of oxytocin receptor binding in the female rabbit forebrain: Variations across the reproductive cycle.

Oxytocin receptors (OTR) have been characterized in the brains of several mammals, including rodents, carnivores, and primates. Their species-specific distribution in the brain has been associated with species differences in social organization, including mating strategy and parenting behavior. In several species, the density of OTR binding in specific brain regions varies according to reproductive condition, including ovarian cycle, pregnancy and lactation. Rabbits are induced ovulators, polygamous, and monoparental but their distribution and regulation of brain OTR has not been described. Here we used receptor autoradiography to quantitatively characterize OTR binding in the brains of estrous, ovariectomized, late pregnant, and lactating does. Intense binding occurred in the prefrontal cortex (PFC), preoptic area (POA), lateral septum (LS; dorsal and ventral), hippocampus, and medial amygdala. Variations among the experimental groups were seen only in PFC, POA, LS. Ovariectomy increased OTR density in PFC but had the opposite effect in POA. Lactating does had significantly reduced OTR density, relative to late pregnancy, in PFC and POA. Our results are consistent with a possible role of OT in modulating social and maternal behavior in rabbits since the brain regions sensitive to OT have been implicated in social interaction, learning and memory, olfactory processing and maternal behavior.

Biostimulation and nursing modify mating-induced c-FOS immunoreactivity in the female rabbit forebrain.

UNLABELLED: Mating in rabbits lasts only 3-5s but profoundly changes the female׳s physiology and behavior (e.g., inhibition of scent-marking and ambulation, changes in EEG, and release of GnRH). The behavioral responsiveness to copulation is reduced in lactating rabbits, relative to estrous does, but is enhanced after suppressing a single nursing bout ("biostimulation"). Little is known about the mechanisms mediating the differential responsiveness to mating among estrous, lactating, and biostimulated rabbits. To begin addressing this issue we quantified the number of c-FOS-immunoreactive (IR) cells in the preoptic area (POA), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), infundibular nucleus (INF), paraventricular nucleus (PVN), supraoptic nucleus (SON), and lateral septum (LS) in mated and unmated does from the above three reproductive conditions. Mating increased c-FOS-IR cells in the POA and PVN relative to unmated estrous does. Biostimulation increased c-FOS-IR cells in the PVN, relative to lactating does, regardless of mating. Lactation reduced the responsiveness of the LS and INF to copulation but increased it in the DMH. No differences were found in the VMH. CONCLUSIONS: a) copulation activates forebrain nuclei that regulate scent-marking (POA), ovulation (INF), and post-coital oxytocin release (PVN); b) lactation and suppression of one nursing bout modulate the magnitude of such changes.

The maintenance and termination of maternal behavior in rabbits: involvement of suckling and progesterone.

Rabbits mated at postpartum estrus become concurrently pregnant and lactating. They wean the first litter shortly before delivering the second one. Lactating-only rabbits do not spontaneously wean their young. These differences suggest that: a) suckling contributes to the long-term maintenance of maternal responsiveness and b) pregnancy-associated factors are essential to promote weaning. To explore if suckling stimulation interacts with the pregnancy condition we compared the behavior of intact and thelectomized (thelx; nipple-removed) rabbits, mated or not at postpartum estrus (Experiment 1). All lactating-only rabbits still showed nursing behavior by postpartum day 44; only 71% of thelx not pregnant does displayed maternal responsiveness for 31days and none by postpartum day 44. If mated at postpartum estrus maternal responsiveness was observed only in 25% of pregnant-lactating does on postpartum day 29 and in 8% of thelx-pregnant rabbits on postpartum day 26. As in pregnant-lactating does progesterone (P) is present across ca. 3weeks and then declines, but it is absent in lactating-only rabbits, in Experiment 2 we explored the effect of injecting P to lactating-only animals on their maternal responsiveness and milk output. P injections (20mg/day) were given across lactation days 1-30 or 1-23. Neither treatment modified maternal behavior: nearly all females entered the nest box, crouched over the litter and suckled it for ca. 3min, as did oil-injected nursing rabbits. In contrast, both P treatments accentuated the decline in milk output, with respect to oil-treated does. Results suggest that suckling promotes the long-term maintenance of maternal behavior while pregnancy-associated factors (not P) are essential to trigger weaning.

Litter size determines circadian periodicity of nursing in rabbits.

Nursing in rabbits occurs inside the nest with circadian periodicity. To determine the contribution of suckling stimulation in regulating such periodicity, we varied the size of the litters provided (1, 2, 4, or 6-8 pups). Nursing does, kept under a 14:10 (L:D) photoperiod, were continuously videotaped from parturition into lactation day 15. Although parturitions occurred throughout the day, a significant negative linear correlation (p < 0.0001; r = -0.68) was evident between time of delivery and time of nursing on lactation day 1, regardless of newborn number: longer intervals between these two events were seen in does delivering in the early morning than in those that gave birth late in the day. In rabbits suckling 6-8 pups, a Rayleigh analysis revealed that the population vector best describing their nursing pattern (across lactation days 1-15) had a phase angle = 58° (corresponding to solar time 0352 h and rho = 0.78; p < 0.001). In contrast, the nursing pattern of does nursing litters smaller than 6 pups did not show circadian periodicity; rather, mothers showed multiple entrances into the nest box throughout the day. Cluster analysis revealed that the main equilibrium point of intervals between suckling bouts shifted from 24 h (6-8 pups) to 6 h (4 and 2 pups) and to as low as 4 h with 1 pup. In the groups nursing 2, 4, or 6-8 pups, most nursing episodes were followed by food and water intake. Those mothers also showed self-grooming of the ventrum and nipples after nursing. The incidence of these behaviors was lower in does nursing 1 pup. In conclusion, nursing in rabbits spontaneously occurs with circadian periodicity, but it is largely modulated by a threshold of suckling stimulation.

Flexibility and adaptation of the neural substrate that supports maternal behavior in mammals.

Maternal behavior is species-specific and expressed under different physiological conditions, and contexts. It is the result of neural processes that support different forms (e.g. postpartum, cycling sensitized and spontaneous maternal behavior) and modalities of mother-offspring interaction (e.g. maternal interaction with altricial/precocious young; selective/non-selective bond). To understand how the brain adapts to and regulates maternal behavior in different species, and physiological and social conditions we propose new neural models to explain different forms of maternal expression (e.g. sensitized and spontaneous maternal behavior) and the behavioral changes that occur across the postpartum period. We emphasize the changing role of the medial preoptic area in the neural circuitry that supports maternal behavior and the cortical regulation and adjustment of ongoing behavioral performance. Finally, we discuss how our accumulated knowledge about the psychobiology of mothering in animal models supports the validity of animal studies to guide our understanding of human mothering and to improve human welfare and health.

New theoretical and experimental approaches on maternal motivation in mammals.

Maternal behavior is expressed in different modalities, physiological conditions, and contexts. It is the result of a highly motivated brain, that allows the female to flexibily adapt her caring activities to different situations and social demands. To understand how mothers coordinate maternal and other motivated behaviors we discuss the limitations of current theoretical approaches to study maternal motivation (e.g. distinction between appetitive and consummatory behaviors), and propose a different approach (i.e. motorically active vs. passive motivations) and a distinction between maternal motivated state and maternal motivated behaviors. We review the evidence supporting dopamine mediation of maternal motivation and describe how different phases of the dopaminergic response - basal, tonic, and phasic release in the nucleus accumbens - relate to increased salience, invigorating behavior, and behavioral switching. The existing and new experimental paradigms to investigate maternal motivation, and its coexpression and coordination with other social or non-social motivations are also analyzed. An example of how specificity of motivational systems (e.g. maternal and sexual behavior at postpartum estrus) could be processed at the neural level is also provided. This revision offers new theoretical and experimental approaches to address the fundamental question of how mothers flexibly adapt and coordinate the different components of maternal behavior with other motivated behaviors, also critical for the survival of the species.

Bimodal binding and free energy of the progesterone receptor in the induction of female sexual receptivity by progesterone and synthetic progestins.

Synthetic progestins (SPs) are used for regulation of fertility, contraception and hormone replacement therapy. The acetylated medroxyprogesterone (MPA), megestrol (MGA) and chlormadinone (CLA) are related to progesterone (P). Other SPs are 19-nortestosterone derivatives such as: norethisterone (NET), norethynodrel (NED) or the 13-ethyl gonane, levonorgestrel (LNG). We studied MPA, NET, NED and LNG in a dose-response manner to induce sexual receptivity in rats. Results showed that MPA, NET and NED act as partial agonists, with similar or lower potency than P. However, LNG is a full agonist. Additionally, the molecules of MPA, MGA, CLA, NET, NED, LNG, and P, were submitted to computer calculations at ab initio quantum mechanics theory, to obtain their electronic structure and molecular properties. The aim was to correlate their behavioral effect with their physicochemical properties. In addition, the crystals of P, NET and LNG bound to the progesterone receptor (PR) were studied. The PR crystallizes as a dimer forming two monomers (mA and mB), in which Gln725 interacts in either of two possible ways with the C3-carbonyl pharmacophore of progestins. P binds differentially to both PR monomers, while NET binds exclusively as mA and LNG binds only as mB in both monomers with no difference. Energetically, binding of LNG and P to mB, is more favorable than that of NET and P to mA. Consequently, this bimodal mechanism increases the action possibilities of SPs on biological systems. Interestingly, progestin potency depends mostly on local molecular structure and electronic features, prevailing over total molecular properties.