Effect of litter separation on 24-hour rhythmicity of plasma prolactin, follicle-stimulating hormone and luteinizing hormone levels in lactating rabbit does
© Cano et al. 2005
Received: 05 April 2005
Accepted: 02 June 2005
Published: 02 June 2005
This work describes the effect of a 48-h litter separation on 24-h patterns of plasma prolactin, FSH and LH concentration in female lactating rabbits kept under a 16:8 light-dark photoperiod (lights on at 0800 h).
Groups of 6–7 female lactating rabbits maintained with their litters or separated from them for 48 h were killed by decapitation on day 11 post-partum, at 6 different time points throughout a 24-h cycle, starting at 0900 h. Plasma levels of prolactin, FSH and LH were measured by specific double antibody radio-immunoassays.
Plasma level of prolactin in control and separated does changed in a similar way throughout the day, showing two maxima, at 0500–0900 h and at 1700–2100 h, respectively. Litter separation significantly augmented plasma FSH and LH and disrupted their 24-h rhythmicity.
Since previous studies had shown that litter separation for short periods of time augmented sexual receptivity and fertility of the doe, the changes in FSH and LH reported may influence the massive release of gonadotropin releasing hormone, LH and FSH triggered by mating or artificial insemination in litter-separated mothers.
In nursing rabbits, sexual receptivity and fertility achieved after artificial insemination is depressed during the period of lactation, presumably through a hormonal antagonism between prolactin and gonadotropin release [1–3]. Several studies have demonstrated that separation of the doe from its litter for short periods of time, prior to artificial insemination, is very effective in stimulating ovarian activity in the mother [4–6], with endocrine changes that may explain the activation of ovarian function [7–9] Thus, separation of the does from their litters could be an effective procedure to augment breeding efficiency under farm conditions [5–7].
It must be noted that the published studies on the endocrine changes taking place in the doe after litter separation have been performed at single time points in the daily cycle, usually in the morning, which is an important drawback in view of the circadian nature of the secretion of the pituitary hormones involved [10, 11]. Indeed a number of circadian functions have been examined in rabbits [12–17], but there is no information on 24-h pattern of hormone release. This prompted us to undertake the present study whose aim was to examine the effect of litter separation for 48 h on 24-h changes in plasma prolactin (PRL), follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels of the doe. Specifically, we sought to answer two questions: (i) did suppression of a major neuroendocrine and circadian stimulus like the stimulation of nipples by the lactating pup affect the 24-h changes in gonadotropin and prolactin release?; (ii) could the changes in circulating hormone levels be related to augmentation of breeding efficiency found after litter separation from the doe?
Materials and methods
The study was performed in 84 multiparous, lactating Californian x New Zealand White crossbreed female doe rabbits. Animals were housed in the research facilities of the Animal Production Department, Universidad Politécnica de Madrid, under controlled light-dark cycles (LD 16:8, light on at 8:00 h), housed in individual metal cages, fed at libitum using a commercial pellet diet (Lab Rabbit Chow, Purina Mills, Torrejón de Ardoz, Madrid, Spain) and having access to tap water ad libitum. The study was performed according to the CEE Council Directives (86/609, 1986) for the care of experimental animals. Groups of 6–7 female lactating rabbits were maintained with their litters or separated from them for 48 h, starting at different times (i.e., at 09:00, 13:00, 17:00, 21:00, 01:00 or 05:00 h). Ninety five percent of the does suckled the pups between 03:30 and 04:30 h during the dark period, as has been previously described . On day 11 post-partum, the does were killed by decapitation at 6 different time points throughout a 24-hour cycle starting at 0900 h. Blood was collected from the cervical wound and the plasma was separated to measure prolactin and gonadotropin concentration.
Plasma prolactin, FSH and LH levels were measured in duplicate samples by specific RIA methods  using AFP-991086, AFP-472176 and AFP-3120489 antibodies for prolactin, FSH and LH respectively, supplied by the National Institute of Health (NIH, Bethesda, MD, USA) and Dr. A. F. Parlow (Harbour-UCLA Medical Center, CA, USA). Hormones were labeled with 125I by the Chloramine T-method . The antibody titers used were 1:62,500 for prolactin, 1:45,000 for FSH and 1:250,000 for LH assays, respectively. The volume of plasma used was 10 μl (prolactin assay), 75 μl (FSH assay) and 100 μl (LH assay). Staphylococcus aureus (prepared by the Department of Plant Physiology, U.A.M., Madrid, Spain) was used to precipitate the bound fraction . The assays were previously validated in our laboratory . All samples were measured in the same assay run to avoid inter-assay variations. The limits of detection for prolactin, FSH and LH were 0.125, 0.48 and 0.05 ng/mL respectively. The intra-assay coefficient of variation, calculated from a pool of plasma measured ten times in the same assay, was < 5%.
After determining that the homogeneity-of-variance assumption was tenable and that the distribution appeared unimodal and nonskewed, the statistical analysis of results was performed by a two-way factorial analysis of variance (ANOVA). Generally, the factorial ANOVA included assessment of the group effect (i.e. the occurrence of differences in mean values between control and separated groups), of time of day effects (the occurrence of daily changes) and of the interaction between the two factors (separation and time, from which inference about differences in timing and amplitude could be obtained). A one-way ANOVA followed by Student-Newman-Keuls' test was then employed to show which time points were significantly different within each experimental group to define the existence of peaks. A Student's t test was performed to assess differences between the experimental groups at particular time intervals. P values lower than 0.05 were considered evidence for statistical significance.
The questions posed in the Introduction may now be answered. First, our results indicate that the 24 h patterns of plasma FSH and LH, but not of prolactin, changed significantly in nursing rabbits after litter separation for 48 h. Second, litter separation disrupted the 24-h rhythmicity of plasma FSH and LH concentration and caused a moderate increase in their concentration (when assessed as the mean 24-h values). Previous studies in the rabbit showed that litter separation for short periods of time augmented sexual receptivity and fertility of the doe [4–9]. Thus, the changes in gonadotropins reported herein could be a reflection of the same mechanisms involved in the massive release of gonadotropin releasing hormone (GnRH), LH and FSH triggered by mating or artificial insemination in litter-separated mothers.
The rabbit exhibits an unusual form of maternal care, with a single and very short visit (3–5 min) every day to nurse . This daily nursing visit of the doe is extremely regular, with some individuals showing a day-to-day variability of only a few minutes. Estrogen, androgen, progesterone and prolactin promote the onset of this behavior in does  while its maintenance relies on stimuli from the litter (i.e., maternal responsiveness is altered or abolished by prevention of mother/young contact at parturition or during early lactation). From a number of studies on the distribution of estrogen, androgen and prolactin receptors, quantification of expression of immediate-early genes, and lesions of structures of the olfactory system, it was concluded that rabbits rely on the same hormonal and extrahormonal factors that stimulate maternal behavior in other mammals except for the very peculiar circadian nursing pattern that is unique to rabbits [22–24].
Since the early observations by McNeilly and Friesen  it is known that postpartum blood levels of prolactin are similar in lactating and postpartum nonlactating females. Such an observation was confirmed in the present study in which plasma prolactin levels, measured at six time intervals in a 24-h cycle (the closest to nursing time was at 05:00 h), were essentially similar in control and litter-separated does. In lactating females, suckling evoked an immediate increase (3- to 5-fold) in circulating prolactin levels, an effect mimicked by the tactile stimulation of the teats . Likewise, in hares, prolactin levels increased significantly during lactation only after suckling stimuli . It must be noted that, in contrast to rabbits, plasma prolactin levels are significantly changed by nursing in most species, the suckling stimulus being an effective masking signal for the 24-h release of prolactin [27, 28]. This does not occur in the doe, the circadian changes of plasma prolactin levels remaining essentially unchanged after litter separation (presumably because of the very short nursing period). This suggests that the circadian secretion of prolactin and the prolactin response to physical stimulation of the nipples are independent phenomena that occur throughout the nursing period.
Previous reports using single sampling procedures  indicated that litter separation decreased thr doe's prolactin levels and did not affect FSH. Discrepancies are possibly dependent on the sampling frequency and time of day examined. Collectively, the results underline the importance of performing 24-h studies to have a more precise picture of the hormonal changes.
Litter separation disrupted the 24-h rhythmicity of both FSH and LH significantly. McNeilly  suggested that a reduction of plasma LH levels found during the light period could be coupled to an increase of pulsatile pattern of hypothalamic GnRH release. In the present study, the does exhibited, after litter separation, an inverse 24-h pattern of LH release with the lowest values during the light phase of daily photoperiod. Presumably, the disrupted 24-h rhythmicity of LH (and FSH) are linked to the greater mating or artificial insemination-induced release of LH and FSH found in does separated from their litters.
In summary, the present study demonstrates the existence of 24-h variations in circulating prolactin, LH and FSH levels in nursing does. Litter separation for short periods of time, an effective procedure to stimulate ovarian activity prior to artificial insemination, markedly influences the secretory patterns of FSH and LH, a finding that can be related to the higher reflex ovulatory response to mating or artificial insemination observed in does separated from their pups. The specific value of the present study in terms of augmenting breeding efficiency should be further explored.
- Theau-Clément M, Roustan A: A study on relationships between receptivity and lactation in the doe and their influence on reproductive performances. J Appl Rabbit Res 1992, 15:412–421.Google Scholar
- Fortum L, Bolet G: Les effects de la lactation sur les performance de reproduction chez la laprine. INRA Prod Anim 1995, 8:44–56.Google Scholar
- Ubilla E, Rebollar PG: Influence of the postpartum day on plasma oestradiol concentrations, sexual behaviour and conception rate, in artificially inseminated lactating rabbits. Anim Reprod Sci 1995, 38:337–344.View ArticleGoogle Scholar
- Pavois V, LeNaour J, Ducep O, Perrin G, Duperray J: A natural method to improve sexual receptivity and fertility in artificially inseminated rabbits. Gémes Journées de la Recherche Cunicole la Rochelle 1994, 2:528–535.Google Scholar
- Alvariño JMR, Del Arco JA, Bueno A: Effect of mother-litter separation on reproductive performance of lactating rabbit female inseminated on day 4 or 11 post partum. World Rabbit Sci 1998, 6:191–194.Google Scholar
- Maertens L: Effect of flushing, mother-litter separation and PMSG on the fertility of lactating does and the performance of the litter. World Rabbit Sci 1998, 6:185–190.Google Scholar
- Castellini C, Canali C, Boiti C: Effect of mother-litter separation for 24-hour by closing the nestbox or change of cage, on rabbit doe reproduction performance. World Rabbit Sci 1998, 6:199–203.Google Scholar
- Ubilla E, Rebollar PG, Pazo D, Esquifino A, Alvariño JMR: Effects of doe-litter separation on endocrinological and productivity varibles in lactating rabbits. Livestock Prod Sci 2000, 67:67–74.View ArticleGoogle Scholar
- Ubilla E, Rebollar PG, Pazo D, Esquifino AI, Alvariño JMR: Endocrine profiles during doe-litter separation and the subsequent pregnancy in rabbits. J Physiol Biochem 2001, 57:23–29.View ArticleGoogle Scholar
- García-Bonacho M, Esquifino AI, Castrillón PO, Reyes Toso C, Cardinali DP: Age-dependent effect of Freund's adjuvant on 24-hour rhythms in plasma prolactin, growth hormone, thyrotropin, insulin, follicle-stimulating hormone, luteinizing hormone and testosterone in rats. Life Sci 2000, 66:1969–1977.View ArticlePubMedGoogle Scholar
- Esquifino AI, Chacon F, Jimenez V, Reyes Toso CF, Cardinali DP: 24-hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone and testosterone in male rats subjected to social isolation. J Circadian Rhythms 2004, 2:1.View ArticlePubMedGoogle Scholar
- Jilge B, Stahle H: The internal synchronization of five circadian functions of the rabbit. Chronobiol Int 1984, 1:195–204.View ArticlePubMedGoogle Scholar
- Jilge B, Hornicke H, Stahle H: Circadian rhythms of rabbits during restrictive feeding. Am J Physiol 1987, 253:R46-R54.PubMedGoogle Scholar
- Jilge B: Restricted feeding: a nonphotic zeitgeber in the rabbit. Physiol Behav 1992, 51:157–166.View ArticlePubMedGoogle Scholar
- Jilge B, Stahle H: Restricted food access and light-dark: impact of conflicting zeitgebers on circadian rhythms of the rabbit. Am J Physiol 1993, 264:R708-R715.PubMedGoogle Scholar
- Jilge B, Hudson R: Diversity and development of circadian rhythms in the European rabbit. Chronobiol Int 2001,18(1):1–26.View ArticlePubMedGoogle Scholar
- Jilge B, Kuhnt B, Landerer W, Rest S: Circadian temperature rhythms in rabbit pups and in their does. Lab Anim 2001, 35:364–373.View ArticlePubMedGoogle Scholar
- Ubilla E, Alvariño JMR, Esquifino AI, Agrasal C: Effects of induction of parturition by administration of a prostaglandin F2 analogue in rabbits: posible modification of prolactin, LH and FSH secretion patterns. Anim Reprod Sci 1992, 27:13–20.View ArticleGoogle Scholar
- Greenwood FC, Hunter WM, Glover JS: The preparation of 131 I labelled human growth hormone of high specific radioactivity. Biochem J 1963, 89:114–123.PubMedGoogle Scholar
- Zarrow MX, Denenberg VH, Anderson CO: Rabbit: frequency of suckling in the pup. Science 1965, 150:1835–1836.View ArticlePubMedGoogle Scholar
- Gonzalez-Mariscal G: Neuroendocrinology of maternal behavior in the rabbit. Horm Behav 2001, 40:125–132.View ArticlePubMedGoogle Scholar
- Gonzalez-Mariscal G, Jimenez P, Beyer C, Rosenblatt JS: Androgens stimulate specific aspects of maternal nest-building and reduce food intake in rabbits. Horm Behav 2003, 43:312–317.View ArticlePubMedGoogle Scholar
- Gonzalez-Mariscal G, Chirino R, Flores-Alonso JC, Rosenblatt JS, Beyer C: Intracerebroventricular injections of prolactin counteract the antagonistic effect of bromocriptine on rabbit maternal behaviour. J Neuroendocrinol 2004, 16:949–955.View ArticlePubMedGoogle Scholar
- Gonzalez-Mariscal G, Chirino R, Rosenblatt JS, Beyer C: Forebrain implants of estradiol stimulate maternal nest-building in ovariectomized rabbits. Horm Behav 2005, 47:272–279.View ArticlePubMedGoogle Scholar
- McNeilly AS, Friesen HG: Prolactin during pregnancy and lactation in the rabbit. Endocrinology 1978, 102:1548–1554.View ArticlePubMedGoogle Scholar
- Caillol M, Mondain-Monval M, McNeilly AS: Pattern of serum concentrations of prolactin and progesterone during pregnancy and lactation in the brown hare (Lepus europaeus). J Endocrinol 1990, 124:11–17.View ArticlePubMedGoogle Scholar
- Chang NG, Nikitovich-Winer MN: Correlation between suckling-induced changes in the ultrastructure of mammotrophs and prolactin release. Cell Tissue Res 1976,166(3):399–406.View ArticlePubMedGoogle Scholar
- McNelly AS: Lactational control reproduction. Reprod Fertil Dev 2001,13(7–8):583–90.View ArticleGoogle Scholar
- McNeilly AS: Lactation and fertility. J Mammary Gland Biol Neoplasia 1997,2(3):291–298.View ArticlePubMedGoogle Scholar
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