Oestrous Rabbits Research Articles

Behavioral and Physiological Differences in Female Rabbits at Different Stages of the Estrous Cycle.

Estrus involves a series of complex physiological signs and changes in behavior before ovulation, which play a crucial role in animal reproduction. However, there have been few studies that evaluate behaviors during the different stages of estrus cycle in female rabbits. Therefore, more detailed information is needed on distinguishing the various stages of the estrous cycle. This study explored the behavioral and physiological differences at various estrous cycle stages in female New Zealand White rabbits. The continuous recording method was employed to record the daily behaviors of twenty postpartum female rabbits during the estrous cycle. Compared with the diestrus stage, the duration of foraging and drinking behavior in estrus decreased significantly, and the frequency of grooming and biting behaviors increased (p < 0.05). Differences in reproductive hormone levels (FSH, LH, P4, and E2) and follicle development were measured at each stage via ELISA and HE staining. The FSH and LH levels showed an increasing trend and then decreased, with the lowest being in late estrus (p < 0.05). The P4 level was the lowest in estrus (p < 0.05), and E2 showed a gradually increasing trend. There was no significant difference in the number of primordial follicles at each stage, but the number of primary follicles in estrus was significantly higher than at the other stages (p < 0.05). To further understand the molecular regulation mechanism of the estrous cycle in female rabbits, we analyzed the ovarian transcription patterns of female rabbits in diestrus (D group) and estrus (E group) employing RNA-seq. A total of 967 differentially expressed genes (DEGs) were screened from the ovaries of female rabbits between the diestrus and estrus groups. A KEGG analysis of DEGs enriched in the estrogen signaling pathway, aldosterone synthesis, and secretion pathway, such as CYP19A1 and IGF1R, was performed. The rabbits' behavior, related physiological hormones, and molecular regulation also differed at different estrous cycle stages. The results provide recommendations for the adequate management practices of postpartum re-estrus and breeding female rabbits.

Oxytocin and vasopressin immunoreactivity in rabbit hypothalamus during estrus, late pregnancy, and postpartum

Mother rabbits construct an elaborate maternal nest before parturition and display a single, brief, daily nursing bout throughout lactation. These features present a unique model for investigating the relevance of changes in neuroendocrine secretion associated with pregnancy and parturition for the regulation of maternal behavior. In the present study we analyzed changes in the location, somal size, and number of oxytocin (OT)and arginine vasopressin (AVP)-immunoreactive (IR) neurons in the hypothalamus of rabbits in estrus, late pregnancy (day 29), and postpartum day 1. From estrus to late pregnancy, the number of OT-IR neurons increased in the scattered cell groups located in the lateral hypothalamic area (LHA), but not in the magnocellular nuclei, i.e., paraventricular nucleus (PVN) and supraoptic nucleus (SON). On postpartum day 1 the increase in the number of OT-IR neurons was sustained in the LHA and became apparent also in the main body of the PVN, in which the number of OT-IR neurons doubled. Increases in the somal size of OT-IR cells were seen in all three nuclei only on postpartum day 1. No OT-IR cells were found in the suprachiasmatic nucleus (SCN). From late pregnancy and into postpartum day 1 increases in the somal size of AVP-IR neurons were detected in the PVN, SON, and LHA but not in the SCN. The number of AVP-IR neurons increased between late pregnancy and postpartum day 1 in the SON only. The changes observed in OT and AVP expression in specific hypothalamic nuclei may be related to specific somatic and behavioral events occurring around the time of parturition, e.g., nest-building, maintenance of homeothermy, elevation of blood volume, and nursing in mother rabbits.

Rabbit sperm function after co-culture with uterine epithelial cells

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.

Characterization of prostaglandin E2 binding to uterine membranes from baboon, rabbit and tree shrew (Tupaia belangeri)

Membrane preparations (100,000 g pellet) of rabbit, baboon and tree shrew (Tupaia belangeri) uteri were studied for binding of [3H]prostaglandin E2 ([3H]PGE2). Unbound [3H]PGE2 was separated by filtration through Whatman GF/F filters. Non-specific binding was determined by the amount of radioactivity associated with the filters in the presence of a 100-fold excess of radioinert PGE2. PGE2 bound to membranes could be displaced by some other prostaglandin (PG) molecules: PGE1, 16,16-dimethyl-PGE2, PGA1 and PGF2 alpha, but not by 6-keto-PGF1 alpha, PGD2 or arachidonic acid. No PGE2 binding was detected using either membrane ghosts from red blood cells or liposomes. Apparent equilibrium of the binding was reached by 60 min. There was no difference in dissociation constant (Kd) values between rabbits of different reproductive stages (mean range +/- S.E.M. was from 4.6 +/- 0.3 to 5.5 +/- 1.0 nM), but pregnant baboons showed a significantly lower value (3.3 +/- 0.4 nM) than did cyclic animals (12.0 +/- 2.0 nM). Binding capacity (Bmax) values, in contrast, were different only between oestrous rabbits and other reproductive stages. The small amounts of Tupaia tissue only permitted estimates of the Kd and Bmax values to be made; these were 3.8 nM and 499 fmol/mg protein for oestrous animals and 5.4 nM and 674 fmol/mg protein for animals on day 7 of pregnancy, assuming only one class of sites. The present results demonstrate the presence of specific binding sites for PGE2 in uteri from several species.

Variations in the mechanical behavior of the rabbit cervix with endocrine state and anatomic site

The mechanical tests of stress-strain and stress-relaxation were done on excised cervical strips from New Zealand rabbits in estrus, less than 10 hours after luteinizing hormone (LH), 10 to 18 hours after LH, 18 to 50 hours after LH, 30 days pregnant, and castrate. The stretch moduli obtained from these tests indicated that a significant softening of the cervix occurred at 10 to 18 hours after LH and during pregnancy. Normally, the internal os is stiffer than the external os, but this difference disappears at 30 days of pregnancy, and both values are lower and identical. The changes in the stretch modulus are not thought to be due to smooth muscle, particularly in the external os, because of the low contractility of this tissue. The conclusion reached is that the rabbit cervix is a good model to use in the investigation of the mechanical behavior of the cervix and may be comparable to the human cervix in physiologic responses.

Effect of ovulation on the ionic and water content of rabbit oviduct.

The water content, extracellular space, intracellular water, potassium, sodium and chloride content of oviduct and uterus removed from rabbits in estrus and 24 and 72 h following hCG injection have been determined. Following ovulation, there was an increase in water content of the region of the oviduct corresponding to the ampullary -isthmic junction which, at 24 h, is probably due to increased intracellular water. In the ampullary-isthmic junction, ovulation decreases the potassium content and, at 24 h in the isthmic region of the oviduct, chloride is reduced. The water and ionic content of mucosa and smooth muscle cells has been calculated and mucosa cells have greater intracellular water and chloride and sodium content and less potassium and extracellular space than the smooth muscle cells of the oviduct. The significance of the changes in ionic and water content is discussed.

Transient responses and continuous behavior of active smooth muscle during controlled stretches.

Controlled length changes were imposed on mesotubarium superius and ovarian ligament smooth muscles from the reproductive tracts of female rabbits in constant estrus. Stretches of up to 35% of the muscle length were applied during isometric contraction, relaxation, and steady-state force levels. Force was continuously monitored and was plotted as a function of length. During constant velocity stretches there was an initial steep rise in force, a rapid downward deviation from the initial slope, and a long region with a constant upward slope. Stretches made during contraction showed smaller initial rises in force and steeper linear portions than did identical comparison stretches made during relaxation. The value of the slope was independent of the prior developed force, but it did depend on whether the muscle was contracting or relaxing. During contraction and steady-state force levels, the slope was independent of the stretch rate, but it was strongly rate dependent during relaxation. Changes in the stretch rate during stretch caused associated changes in muscle force; the relationship was curvilinear and was exaggerated during relaxation. The findings are placed in the context of a sliding-filament--cross-bridge hypothesis.

Oviduct acid mucus glycoproteins in the estrous rabbit: ultrastructure and histochemistry.

Epithelial glycoproteins are likely to be important in many aspects of reproduction. The rabbit oviduct produces mucus glycoproteins. This is indicated both by histochemistry and by gelation of a mucus coat around the rabbit ovum during its tubal transport. We report here that the production of highly acid mucus glycoprotein (apparently of the type that coast the ovum) is confined to the isthmus and, to a lesser extent, the mucosal crypts of the ampullary-isthmic junction; the ampulla is not involved. Using a method of perfusion-fixation that includes the polycation alcian blue in conjunction with glutaraldehyde to precipitate and stabilize glycoproteins, we have demonstrated that this mucus, at least in rabbits in estrus, occupies the isthmic lumen but not the ampullary lumen. Histochemistry shows that it is the electron-lucent secretory granules of the isthmus and ampullary-isthmic junction, but not the denser granules of the ampulla, that exhibit staining characteristics of highly-acid mucus glycoproteins. Important opportunities are likely to exist for interaction of this isthmic mucus with spermatozoa and with fertilized ova during their isthmic transport.

Ultrastructure of rabbit endosalpinx and glycoprotein pattern of the oviduct fluid in the presence of copper intrauterine devices.

Oviduct fluid was collected from 24 mature female rabbits in estrus fitted with a copper intrauterine device (IUD) in one of the uterine horns by laparotomy and examined for total sugar and protein content. The percentage of total sugar in the dry protein-containing material from the experimental side was 7.3 compared to 5.2 from the control side. The percentage of total protein was 67.0% of the total dry material from the experimental side, compared to 77.7% in the control side. Cellulose acetate electrophoresis confirmed these findings, demonstrating an increase in the strength of the periodic acid-Schiff stain bands of the glycoproteins from the copper IUD side and a weaker Ponceau-S stain of the protein bands. The ampullary-isthmic area and the uterotubal junction of both oviducts were examined with the scanning electron microscope. The secretory and ciliary cells on the copper IUD side were damaged in these two areas. These changes can be attributed to the presence and action of the copper ions in the oviduct milieu.

Prostaglandins and follicular functions.

Ovulation The involvement of prostaglandins (PGs) in the regulation of ovarian follicular function was first postulated on the basis of the demonstration that inhibitors of prostaglandin synthesis, such as aspirin and indomethacin, were capable of blocking ovulation in rats (Armstrong & Grinwich, 1972; Orczyk & Behrman, 1972). These initial findings were soon confirmed in several other species, including mice (Lau, Saksena & Chang, 1974), rabbits, (Grinwich, Kennedy & Armstrong, 1972; O'Grady, Caldwell, Auletta & Speroff, 1972), rhesus and marmoset monkeys (Wallach, de la Cruz, Hunt, Wright & Stevens, 1975; Maia, Barbosa & Coutinho, 1978), pigs (Ainsworth et al., 1979) and goldfish (Stacey & Pandey, 1975). In two of these species (rabbits and goldfish), the inhibitor was effective when applied locally to the follicle, indicating that the blockade was exerted directly upon the follicle, rather than being mediated via some indirect mechanism, such as through inhibition of gonadotrophin secretion. Further evidence of a role of prostaglandins at the follicular level was provided by the findings that intrafollicular levels of prostaglandins of both the E and F series increased markedly in several of these species shortly before ovulation (Yang, Marsh & LeMaire, 1974; Armstrong, Moon & Zamecnik, 1974; Bauminger & Lindner, 1975; Ainsworth, Baker & Armstrong, 1975; Tsang, Ainsworth, Downey & Armstrong, 1979a); indomethacin, at dosages which prevented ovulation, effectively prevented these increases. The observation that injection of antiserum against PGs blocked the LH-induced ovulation in oestrous rabbits, whether administered systemically (Lau et al., 1974) or via intrafollicular injection (Armstrong et al., 1974), added support to the concept of a role of prostaglandins in ovulation. Antiserum to PGF-2α appeared to be more effective than that to PGE in these experiments, suggesting that PGF-2α was the prostaglandin of greater importance in ovulation.

Effect of copper intrauterine devices on the chemical composition of rabbit oviduct fluid.

Oviduct fluid, collected from 65 mature, female rabbits in estrus and in which a modified copper intrauterine device (IUD) was introduced by laparotomy in one of the uterine horns, was examined for chemical composition. No differences were noted for sodium, potassium, calcium, chloride and bicarbonate between the two sides. The copper content was found to be significantly higher in the IUD side (12.80 +/- 3.01 microgram/100 ml), as compared to the control side (9.0 +/- 1.0 microgram/100 ml), the toxicity of which, to blastocyst development, has been reported in some cases. Total protein in the experimental side was significantly higher (258.52 +/- 36.22 mg/100 ml) than in the control side (156.67 +/- 26.45 mg/100 ml). The same was found for sialic acid, 5.30 +/- 0.62 microgram/ml, compared to 3.66 +/- 0.27 microgram/ml, suggesting that a part of the increased protein can be ascribed to glycoprotein. Oviduct fluid glucose was found to be lower in the copper IUD side than in the control side (10.5 +/- 0.2 vs. 17.7 +/- 0.3 mg/100 ml). These findings may be explained on the basis of an inflammatory process, taking place in the side of the copper IUD, and should be used with cautiousness in adolescent, and nulligravidae women.

The cytosol receptors for progesterone in the different parts of rabbit fallopian tube and uterus during ovum transport

Progesterone receptors were determined in the cytosol from the ampulla, ampullary- isthmic junction and isthmus of rabbit fallopian tube and uterus of estrus and pregnant rabbits. The receptor levels when compared among its various anatomical segments, were the same in ampulla, isthums and uterus but maximum in ampullary-isthmic junction. Significant differences were observed in mated animals at 14, 24, 34, 48, 70 and 144 h after coitus. The receptor concentrations in portions of the fallopian tube showed no significant change between 14 and 24 h after coitus, except for a decrease in ampullary-isthmic junction at 24 h. At 34 h the concentration of receptor further decreased in all parts of the tube. At 48 and 70 h after coitus, receptor concentrations decreased gradually in ampulla and ampullary-isthmic junction, while isthmus showed a gradual increase. At 144 h, the receptor concentration showed no further change in ampulla and ampullary-isthmic junction; however, isthmus showed a decline. The uterine receptor concentration declined steadily from estrus till 70 h after coitus, however, it was increased at 144 h. The dissociation constant (Kd) of cytosol receptor in all the tissues at estrus and during early pregnancy was found similar. The implications of these changes in relation to the normal ovum transport have been correlated in this paper.

Incorporation of [U-14C]glucose in vitro by the various parts of the fallopian tube and uterus of the rabbit during ovum transport.

Incorporation of [U-14C]glucose in vitro was determined in ampullary, ampullary-isthmic junction and isthmic segments of the Fallopian tube and uterus of intact (oestrous) rabbits and also at various times after mating. In the oestrous animals, incorporation was slightly higher in the ampullary segment compared with the isthmic and the ampullary-isthmic junction and finally the uterus. The pattern changed significantly in the pregnant animals. At 14 and 24 h post coitum, the pattern of incorporation was similar to that found at oestrus except for an increase in uterine incorporation. However, at 48 and 72 h post coitum, incorporation by the isthmic segment and the uterus was significantly increased compared with corresponding values at oestrus, and 14 and 24 h post coitum. The implication of these changes during egg transport through the Fallopian tube is discussed.

Failure of histamine to induce the release of luteinizing hormone in castrated rats primed with sex steroids.

Instituto de Investigaciones Cerebrales, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina (Received 10 March 1978) Injection of histamine into the third ventricle causes the release of luteinizing hormone (LH) in ovariectomized, steroid-primed and pro-oestrous rats (Donoso, Banzan &amp; Borzino, 1976; Donoso, 1978). In oestrous rabbits, intraventricular injection of large doses of histamine or injection of histamine into the mediobasal hypothalamus induces ovulation and secretion of progesterone by the ovaries (Sawyer, 1955; Endröczi &amp; Hilliard, 1965). However, histamine has no effect on the concentration of LH in the plasma of male rats (Donoso &amp; Banzan, 1976). Together, these results suggest that histamine is able to induce the release of LH both before ovulation and in the presence of an adequate hormonal background. The positive feedback effects of oestradiol and progesterone on the release of gonadotrophins are absent in male rats. Injection of oestradiol, progesterone or testosterone into steroid-primed

Evidence of a uteroglobin-like protein in epithelial cells of reproductive and non-reproductive tissues of the rabbit

An antiserum raised in a goat to a uteroglobin-like protein isolated from uterine fluid of oestrous rabbits was used in an immune fluorescence test to localize an antigen present in the reproductive tract of oestrous and pseudopregnant rabbits and mammary gland tissue. The antigen was also present in the vas deferens and seminal vesicle, but not in testis. Non-reproductive tissues, such as lung, small intestine, bladder and thyroid showed specific fluorescent staining which was eliminated or significantly reduced by absorption of the antiserum with a purified uteroglobin preparation.

Effect of progesterone on adrenoceptors in the isthmus of the rabbit oviduct

The effect of adrenergic agonists on the contractility of circular muscle of the isthmus of the oviduct was studied in isolated tissues from rabbits in estrus and after progesterone pretreatment. α-Adrenoceptor sensitivity was not altered by progesterone, and the order of potencies of α-agonists was similar to that for α-adrenoceptors in other rabbit tissues. Progesterone pretreatment increased the maximal inhibitory effect of isoprenaline on β-adrenoceptors, but did not alter sensitivity to the agonist, suggesting that there had been an increase in the number of β-adrenoceptors rather than a change in their affinity. The order of potencies of β-agonists was similar to that for the β 1-adrenoceptor in other rabbit tissues.

The effects of prostaglandin and mating on release of LH in the female rabbit.

The release of LH in response to prostaglandin (PG) treatment of female rabbits in various reproductive states was compared with the surge following mating. Intracarotid infusion of PGE-2 or PGF-2alpha (0-3--900 microgram/h) into non-receptive and pseudopregnant does resulted in small, 2--4-fold elevations in jugular vein LH concentration. Similar doses of PGF-2alpha in oestrogen-pretreated, oestrous does stimulated a 13-fold increase in plasma LH levels. Mating resulted in a much larger release of LH, as plasma levels increased approximately 60-fold from 1-1 +/- 0-2 (S.E.M.) ng/ml to 67-8 +/- 10-5 ng/ml. These results indicate that PG can stimulate the hypothalamic-hypophysial axis to release LH in non-receptive, pseudopregnant and oestrogen-pretreated, oestrous rabbits.

Effect of IUDS on uterine cyclic AMP in rabbits

The mechanism(s) whereby an intrauterine device (IUD) prevents implantation are poorly understood (Eckstein, 1970). Sim (1974) showed that the presence of an IUD in the rat uterus was associated with an increase in cyclic AMP content and adenyl cyclase activity. Cyclic AMP has been implicated in a variety of uterine biosynthetic processes (Hechter, Yoshinaga, Halkerston & Birchall, 1967) and in the control of spontaneous uterine contractions (Johansson & Anderson, 1975). The present study was designed to determine the effect of a plastic and copper-bearing IUD on levels of cyclic AMP in the uteri of oestrous rabbits.

Cumuli Oophori in Tissue Culture: Hormone Production, Ultrastructure, and Morphometry of Early Luteinization

The steroidogenic activity of cumulus oophorus granulosa cells obtained from unstimulated rabbits in estrus has been investigated at closely timed intervals in short-term cultures. The presence of viable, structurally well preserved oocytes and the addition of large follicle follicular fluid had no apparent inhibitory effect on progesterone or estrogen synthesis and secretion. Progesterone secretion was especially stimulated by the addition of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the culture medium. A similar increment was also seen, although inconsistently, for estrogen secretion. Ultrastructural evidence of luteinization was demonstrated by quantitative morphometric techniques in control cultures as early as 6 hours after explantation and was shown to relate well to biochemical luteinization. It is suggested that the main modifications in cytoplasmic organelles concomitant with luteinization may be represented only by volume and surface changes rather than by alterations in morphology. Therefore, such changes need to be assessed quantitatively. Oocytes exhibited isolated maturative changes almost exclusively in FSH-LH cultures, such as cortical granule synthesis, resumption of meiosis, polar body extrusion, and two-cell parthenogenetic cleavage. It is suggested that cumulus granulosa cells may play a significant role in the resumption of "spontaneous" maturation in oocytes after their release in vitro from their follicles. Characteristic, thin-walled vesicles, either isolated or in rosette-like formation with cumulus granulosa cells, were described. A possible common nature was suggested for these vesicles and the so-called "Call-Exner bodies."

Effect of Luteinizing Hormone and Cyclic Adenosine 3',5'-Monophosphate on Steroidogenesis in the Ovarian Follicle of the Rabbit

Mature ovarian follicles have been isolated from estrus rabbits and the effects of gonadotropin and cyclic AMP on steroidogenesis in this tissue determined. Gonadotropins used include LH, FSH, and prolactin; follicular levels of progesterone, 17-hydroxyprogesterone, testosterone and 17beta-estradiol in all experiments were quantitated by radioimmunoassay. Incubation of follicles with LH in concentrations ranging from 0.05 to 50 mug/ml medium yielded increases in the total ng steroid/follicle. Five mug LH/ml gave a maximal response with no further increase in steroid concentration when the LH was raised to 50 mug/ml. Prolactin had no effect on follicular steroidogenesis while the stimulatory action of a FSH preparation could only be partially attributed to LH contamination. Cyclic AMP also proved to be a potent stimulatory agent in follicular steroidogenesis with maximal increases at 20 mumoles/ml and a decline in the ng steroid/follicle when cyclic AMP was raised to 30 and 40 mumoles/ml. The effects of LH and cyclic AMP proved to be nonadditive; incubation of follicles simultaneously with 5 mug LH/ml and 20 mumoles cyclic AMP/ml yielded steroid concentrations which were no different than levels following incubation with either agent alone. Taken together, these results demonstrate that both LH and cyclic AMP cause increases in radioimmunoassayable steroid in the rabbit follicle in vitro and that LH probably acts by way of a cyclic AMP intermediate.