Multiparous Rabbits Research Articles

Differential ovulation responses in nulliparous and multiparous rabbit females: A cannula-mediated study

In order to explore a less stressful and hormone-independent insemination method in rabbits, we aimed to evaluate the influence of a new insemination cannula on reproductive performance. Two assays were conducted. In the first, 136 nulliparous and multiparous females were treated with 12.5 IU of eCG to improve their receptivity and divided into four stimulation groups (aGnRHControl, Extender, Seminal and Sperm) to test ovulation frequency and rate. The control group received 1 µg of buserelin acetate intramuscularly to induce ovulation. The other groups used a 3D printed cannula to introduce 0.5 ml of extender, undiluted seminal plasma or resuspended sperm into the vagina. After six days, laparoscopy was performed to assess ovulation frequency and rate. Results showed that nulliparous does exhibited higher ovulation frequencies compared to multiparous does, regardless of the stimulation group.In the second assay, reproductive performance (delivery rate and litter size at birth) of 109 females treated with 12.5 IU of eCG to improve their receptivity was studied after insemination with a seminal dose using the 3D printed cannula. Forty-nine of them were induced to ovulate with buserelin acetate as a control. The results confirmed previous findings, as cannula stimulation allowed better delivery rates in receptive nulliparous than multiparous does. Differential response to multiparous does to the physical stimulus of the cannula could be supported by the histological changes and loss of contractile response that occur after multiple births. Litter sizes at birth were similar between experimental groups.In conclusion, the 3D designed cannula showed promising results in nulliparous does in ovulating frequency, and further refinement is needed to enhance its effectiveness, particularly in multiparous rabbits.

Shift from Pro- to Anti-Inflammatory Phase in Pelvic Floor Muscles at Postpartum Matches Histological Signs of Regeneration in Multiparous Rabbits.

Background and Objectives: Pelvic floor muscles (PFM) play a core role in defecation and micturition. Weakening of PFM underlies urogynecological disorders such as pelvic organ prolapse and stress urinary incontinence. Vaginal delivery damages PFM. Muscle trauma implies an inflammatory response mediated by myeloid cells, essential for subsequent recovery. Molecular signaling characterizing the pro-inflammatory phase shifts M1 macrophages to M2 macrophages, which modulate muscle repair. The present study aimed to evaluate histological characteristics and the presence of M1 and M2 macrophages in bulbospongiosus (Bsm) and pubococcygeus muscles (Pcm). Materials and Methods: Muscles from young nulliparous (N) and multiparous rabbits on postpartum days three (M3) and twenty (M20) were excised and histologically processed to measure the myofiber cross-sectional area (CSA) and count the centralized myonuclei in hematoxylin-eosinstained sections. Using immunohistochemistry, M1 and M2 macrophages were estimated in muscle sections. Kruskal-Wallis or one-way ANOVA testing, followed by post hoc tests, were conducted to identify significant differences (p < 0.05). Results: The myofiber CSA of both the Bsm and Pcm of the M3 group were more extensive than those of the N and M20 groups. Centralized myonuclei estimated in sections from both muscles of M20 rabbits were higher than those of N rabbits. Such histological outcomes matched significant increases in HLA-DR immunostaining in M3 rabbits with the CD206 immunostaining in muscle sections from M20 rabbits. Conclusions: A shift from the pro- to anti-inflammatory phase in the bulbospongiosus and pubococcygeus muscles of multiparous rabbits matches with centralized myonuclei, suggesting the ongoing regeneration of muscles.

Liquid crystal elastomer based dynamic device for urethral support: Potential treatment for stress urinary incontinence

Stress urinary incontinence (SUI) is characterized by the involuntary loss of urine due to increased intra-abdominal pressure during coughing, sneezing, or exercising. SUI affects 20–40% of the female population and is exacerbated by aging. Severe SUI is commonly treated with surgical implantation of an autologous or a synthetic sling underneath the urethra for support. These slings, however, are static, and their tension cannot be non-invasively adjusted, if needed, after implantation. This study reports the fabrication of a novel device based on liquid crystal elastomers (LCEs) capable of changing shape in response to temperature increase induced by transcutaneous IR light. The shape change of the LCE-based device was characterized in a scar tissue phantom model. An in vitro urinary tract model was designed to study the efficacy of the LCE-based device to support continence and adjust sling tension with IR illumination. Finally, the device was acutely implanted and tested for induced tension changes in female multiparous New Zealand white rabbits. The LCE device achieved 5.6% ± 1.1% actuation when embedded in an agar gel with an elastic modulus of 100 kPa. The corresponding device temperature was 44.9 °C ± 0.4 °C, and the surrounding agar temperature stayed at 42.1 °C ± 0.4 °C. Leaking time in the in vitro urinary tract model significantly decreased (p < 0.0001) when an LCE-based cuff was sutured around the model urethra from 5.2min ± 1min to 2min ±0.5min when the cuff was illuminated with IR light. Normalized leak point force (LPF) increased significantly (p = 0.01) with the implantation of an LCE-CB cuff around the bladder neck of multiparous rabbits. It decreased significantly (p = 0.023) when the device was actuated via IR light illumination. These results demonstrate that LCE material could be used to fabricate a dynamic device for treating SUI in women.

Targeted neuromodulation of pelvic floor nerves in aging and multiparous rabbits improves continence

Pelvic floor muscle stretch injury during pregnancy and birth is associated with the incidence of stress urinary incontinence (SUI), a condition that affects 30–60% of the female population and is characterized by involuntary urine leakage during physical activity, further exacerbated by aging. Aging and multiparous rabbits suffer pelvic nerve and muscle damage, resulting in alterations in pelvic floor muscular contraction and low urethral pressure, resembling SUI. However, the extent of nerve injury is not fully understood. Here, we used electron microscopy analysis of pelvic and perineal nerves in multiparous rabbits to describe the extent of stretch nerve injury based on axon count, axon size, myelin-to-axon ratio, and elliptical ratio. Compared to young nulliparous controls, mid-age multiparous animals showed an increase in the density of unmyelinated axons and in myelin thickness in both nerves, albeit more significant in the bulbospongiosus nerve. This revealed a partial but sustained damage to these nerves, and the presence of some regenerated axons. Additionally, we tested whether electrical stimulation of the bulbospongiosus nerve would induce muscle contraction and urethral closure. Using a miniature wireless stimulator implanted on this perineal nerve in young nulliparous and middle age multiparous female rabbits, we confirmed that these partially damaged nerves can be acutely depolarized, either at low (2–5 Hz) or medium (10–20 Hz) frequencies, to induce a proportional increase in urethral pressure. Evaluation of micturition volume in the mid-age multiparous animals after perineal nerve stimulation, effectively reversed a baseline deficit, increasing it 2-fold (p = 0.02). These results support the notion that selective neuromodulation of pelvic floor muscles might serve as a potential treatment for SUI.

Multiparity affects conduction properties of pelvic floor nerves in rabbits.

IntroductionWomen often develop pelvic floor dysfunction due to damage to the pelvic musculature during childbirth; however, the effect on pelvic floor nerves function is less understood. This study used adult rabbits to evaluate the electrophysiological and histological characteristics of the bulbospongiosus (Bsn) and pubococcygeus nerves (Pcn) in multiparity.MethodsCompound nerve action potentials (CNAP) were compared between age‐matched nulliparous and multiparous animals and associated to the histological characteristics of myelinated axons from the Bsn and Pcn nerves. The extensor digitorum longus nerve (EDLn) was used as negative control. Data were analyzed with unpaired two‐tailed Student's t test or Mann–Whitney U test to determine significant differences between groups.ResultsThe onset and peak latencies, duration, and conduction velocity of the motor fibers in these pelvic nerves were not significantly different between nulliparous and multiparous animals. However, the peak‐to‐peak amplitude and area of the CNAP in both Bsn and Pcn were reduced in multiparous rabbits. Histology showed a higher percentage of axons with myelin disorganization caused by multiparity in these pelvic nerves. Together, the data indicate a reduction in the number of functional pelvic axons due to multiparity. As expected, no effect of parity was observed in the EDLn controls.ConclusionsPresent findings demonstrated that multiparity affects myelination and consequently conduction properties in the small pelvic floor nerves.

Co-relation of estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female laboratory rabbits.

This study was designed to investigate the relationship between the estrous cycle phases with uterine bacterial and fungal flora in non-pregnant female rabbits. Thirty laboratory mature multiparous rabbits were used for this purpose. Samples from uterine lavage for culture of bacteria and fungi were collected at different stages of estrous cycle (based on vaginal cytology), and histopathological observations were evaluated based on the scoring system used for defining the infection of the uterus. Various types of bacteria and fungi were isolated from rabbits at all stages of estrous cycle. The widest variety of bacteria and fungi was isolated at Di-estrous stage and the lowest variety was detected at estrous stage. Klebsiella oxytoca as well as yeast have been isolated at all stages of estrous cycle. This study showed that infection with K. oxytoca and yeast had no relationship with different stages of estrous cycle but other bacteria and fungus were associated with one or more stages of the estrous cycle in rabbits.

Multiparity modifies contractile properties of pelvic muscles affecting the genesis of vaginal pressure in rabbits.

To characterize the contractile properties of the bulbospongiosus (Bsm), isquiocavernosus (Ism), and pubococcygeus muscles (Pcm), and their involvement in the genesis of vaginal pressure in nulliparous and multiparous rabbits. Age-matched nulliparous and multiparous rabbits were used to record the isometric contractile responses of each muscle as well as the intravaginal pressure evoked by single square electrical pulses and stimulation trains of ascending frequency. To establish significant differences between groups, two-tail unpaired Student t tests were carried out. The linear correlation between intravaginal pressure and muscle contractile force was analyzed with Pearson correlation tests. For all cases, a P ≤ 0.05 was set as statistically significant. Multiparity decreased the contractile force of Bsm and Ism generated by high-frequency stimulation trains. The normalized force of the Pcm increased when evoked at 1, 4, and 10 Hz while this decreased at higher frequencies (20, 50, and 100 Hz). The contraction of both Bsm and Ism raised particularly the pressure on the perineal vagina while that of the Pcm increased the pressure in the pelvic vagina. Such a functional segregation is still present in multiparous rabbits albeit it was modified. Multiparity induces changes in the contractile responses of Bsm, Ism, and Pcm, which alterates the vaginal pressure.

Fiber type composition of pubococcygeus and bulbospongiosus striated muscles is modified by multiparity in the rabbit.

We analyzed the effect of multiparity on the fiber type composition of two skeletal muscles involved in the maintenance of the micturition process, the pelvic pubococcygeus (Pc) and perineal bulbospongiosus (Bs) muscles in nulliparous and multiparous rabbits (Oryctolagus cuniculus). We used the basic ATPase and NADH-TR techniques to identify and characterize slow, intermediate, and fast fiber types and glycolitic and oxidative fibers in muscles, respectively. Pc muscles of multiparous rabbits present relatively high percentages of slow and intermediate fibers but a low percentage of fast fibers (P < 0.05) as compared to Pc muscles from nulliparous rabbits, while percentages of glycolytic and oxidative fibers were similar (P > 0.05). Bs muscles of multiparous rabbits had a higher proportion of intermediate and glycolytic fibers (P < 0.05) than muscles of nulliparous. Both, Pc and Bs muscles of nulliparous and multiparous rabbits contain slow fibers with similar large cross sectional area, but fast fibers in multiparous muscles showed small cross sectional area than in nulliparous. Multiparity modified the fiber type composition of Pc and Bs muscles in female rabbits. We propose that the contractile force and the physiological role of both muscles during micturition are affected because of the observed changes in the relative composition of muscle fiber types.

Tissue alterations in urethral and vaginal walls related to multiparity in rabbits.

In rodents, vaginal distention after delivery or experimental manipulation affects innervations as well as the amount of striated/smooth musculature or collagen in both the urethra and vagina. These changes are associated with modifications in excretory and reproductive processes. Although successive and consecutive vaginal deliveries (multiparity) affect the contractile and functional properties of the female lower urogenital tract (LUT), its impact on LUT morphometry, including persistency, has been barely studied. The caudal urethra (CU) and cranial (V1) and caudal (V2) pelvic vaginal regions were excised from young and adult nulliparous (YN and AN, respectively) and multiparous (YM and AM, respectively) rabbits. Tissues were histologically processed and stained with Masson's trichrome. The thickness of the tissue layers and areas covered by tissue components were measured and compared using two-way ANOVA followed by Student-Newmann-Keuls tests to determine statistical differences (P ≤ 0.05). Compared to YN, YM, and AN tissues showed a reduction in the thickness of the epithelium, as well as in areas covered by striated musculature, collagen, and blood vessels of the LUT. In comparison with YM, only some morphometric changes were recovered in the AM group. Our study shows that multiparity and age can be associated with epithelial and muscular atrophy of urethral and vaginal walls. The morphometry of the LUT between young and adult female rabbits varies with multiparity. These findings may help to better understand the effects of multiparity on young and adult females and its correlation with the development of pelvic dysfunctions.

Effect of multiparity on morphometry and oestrogen receptor expression of pelvic and perineal striated muscles in rabbits: is serum oestradiol relevant?

ObjectiveTo determine changes in morphometry and expression of oestrogen receptors (OR) in the pubococcygeus and bulbospongiosus muscles, and the concentration of serum oestradiol associated with multiparity. Study designTwelve chinchilla-breed female rabbits were divided into multiparas who had undergone four consecutive deliveries and age-matched virgin nulliparas. Pubococcygeus and bulbospongiosus muscles were surgically removed from each rabbit and processed histologically. Fibre cross-sectional area, number of peripheral nuclei, and expression of ORα and ORβ were measured for each muscle. Serum samples were obtained and the concentration of serum oestradiol was quantified. ResultsMultiparity increased (p≤0.05) fibre cross-sectional area and the number of peripheral nuclei per fibre in pubococcygeus muscle, but not in bulbospongiosus muscle. Expression of both ORα and ORβ was high (p≤0.05) in both muscles from multiparous rabbits. A rise in serum oestradiol was measured at the end of the second pregnancy, which was absent (p≤0.05) at the end of the fourth pregnancy. The concentration of serum oestradiol was similar (p>0.05) in nulliparous and multiparous rabbits. ConclusionsMultiparity caused morphometric changes in pubococcygeus muscle but not in bulbospongiosus muscle. As OR expression was high for both muscles, some properties related to fibre composition or muscle physiology could be affected. The finding that serum oestradiol was not elevated at the end of the fourth pregnancy could be related to changes in pelvic and perineal muscles associated with multiparity.

Multiparity Causes Uncoordinated Activity of Pelvic- and Perineal-Striated Muscles and Urodynamic Changes in Rabbits

Temporal and coordinated activation of pelvic- (pubococcygeous) and perineal- (bulbospongiosus and ischiocavernosus) striated muscles occurs during micturition in female rabbits. We have hypothesized that the coordinated activation of pelvic and perineal muscles is modified during the micturition of young multiparous rabbits. Young virgin and multiparous female chinchilla rabbits were used to simultaneously record cystometrograms and electromyograms of the pubococcygeous, ischocavernosus, and bulbospongiosus muscles. Bladder function was assessed using standard urodynamic variables. The temporal coordination of pelvic- and perineal-striated muscle activity was changed in multiparous rabbits. The cystometrogram recordings were different than those obtained from virgin rabbits, as seen in alterations of the threshold volume, the residual volume, the voiding duration, and the maximum pressure. In rabbits, we find that multiparity causes uncoordinated activity of pubococcygeous, ischiocavernosus, and bulbospongiosus muscles and modifies the urodynamics.

Interaction of reproduction rhythm, suckling and parity on mating incidence, ovarian dynamics and estrogensecretion in the rabbit doe

The effects of suckling and remating interval (11 or 21 d post partum) were studied in primiparous (P=25) and multiparous (M=23) hybrid rabbits. In P rabbits, suckling had no significant effect on mating incidence; however, a higher percentage of females accepted the male on d 11 than on d 21 (P<0.01). In M rabbits, no significant differences in mating performance due to either day, suckling or their interaction were found. A higher proportion of nonsuckled than suckled P rabbits ovulated (P<0.01). Ovulation incidence was not affected significantly by day of mating, suckling or their interaction in M rabbits. A comparison of the day influence in P and M rabbits on mating incidence showed a greater effect in the P (P<0.05) while the suckling effect on ovulation performance showed an insignificant difference in the two parity groups. Ovulation rate, as assessed by ultrasound, was not influenced significantly by day or suckling in either P or M rabbits. The follicle distribution was marked by fewer large follicles and lower plasma estrogen levels were detected in suckled than in nonsuckled P and M rabbits (P<0.05). Further research is needed to elucidate these phenomena also when AI is performed.

Effect of some factors on the fusion rate of bovine–rabbit interspecies reconstructed eggs

AbstractTests were conducted to highlight the effects of donor cell treatment (bovine G0- and non-G0 phase somatic cells refrigerated for 1–5 or 12–14 days at 4°C), fusion solution (with or without cytochalasin B) and age of the rabbit (young, 3–5-months old; or multiparous, 10–12 months old) as oocyte donor on the fusion and mortality rates of bovine–rabbit interspecies reconstructed eggs. Results showed that there were no significant differences (P&gt;0.05) in fusion and mortality rates of eggs reconstructed with G0 cells or non-G0 cells as nuclear donor somatic cells. The same was also true when nuclear donor somatic cells (G0 cells or non-G0 cells) were treated at low temperature (4°C) for 1–5 or 12–14 days. The fusion rate of reconstructed eggs did not change significantly (P&gt;0.05) when adding cytochalasin B into the fusion solution, but the mortality rate increased significantly (P&lt;0.05). No significant effect (P&gt;0.05) was found in fusion rate of the eggs reconstructed with young rabbits' and multiparous rabbits' oocytes as recipient, but the mortality rate decreased significantly (P&lt;0.01) for young rabbit oocytes compared with multiparous rabbit ones.

Gene expression in rabbit menisci during pregnancy

Recent studies have indicated that pregnancy can affect cellular activity in connective tissues such as cartilage, ligaments, and tendons. However, the impact of pregnancy on cellular activity in the menisci, a critical component in joint function, has not been reported. Therefore,the purpose of this study was to evaluate mRNA levels for several relevant molecules in both medial and lateral menisci from the knees of first-time pregnant immature rabbits (primigravida), third-time pregnant mature rabbits (multiparous), and nonpregnant rabbits (age-matched immature and mature controls) by the sensitive molecular technique of semiquantitative reverse transcription--polymerase chain reaction. Total RNA yields from the medial meniscus of multiparous rabbits were reduced to 66% of age-matched control values; however, yields from medial and lateral menisci from primigravida animals or the lateral meniscus of multiparous animals were not significantly depressed. DNA yields were not affected by pregnancy. Type I collagen mRNA levels were significantly depressed in both menisci only in primigravida rabbits. Versican mRNA levels were significantly elevated in both menisci only in multiparous rabbits. None of the transcripts for the other matrix molecules assessed were influenced by pregnancy. Collagenase mRNA levels were unaffected by pregnancy, but TIMP-1 mRNA levels were significantly decreased in the medial meniscus of primigravida rabbits and in the lateral meniscus of multiparous rabbits. Complex changes in the pattern of mRNA expression were observed for growth factors (TGF-beta bFGF, and IGF2). Inducible nitric oxide synthase mRNA expression was not altered during pregnancy. Transcripts for cyclooxygenase-2 were 61 and 50% of control values in the medial meniscus of primigravida and multiparous animals, respectively, while cyclooxygenase-2 mRNA levels were 150 and 188% of control values in the lateral meniscus from primigravida and multiparous animals, respectively. Such results indicate that pregnancy-induced changes in the pattern of mRNA expression are meniscus specific. Furthermore, the results support the hypothesis that different connective tissues of the knee respond to pregnancy in a unique manner.

Influence of pregnancy on gene expression in rabbit articular cartilage

Objective: Articular cartilage is known to be influenced by estrogen and the pregnancy-associated hormone, relaxin,in vitro. Such observations have raised the possibility that articular cartilage in females may be subjected to unique regulatory influences by such hormonesin vivo. The purpose of this study was to evaluate mRNA levels for several relevant molecules in the articular cartilage of pregnant and non-pregnant rabbits.Design: Total RNA was extracted from New Zealand White rabbit knee articular cartilage using the TRIspin method. The total RNA was reverse transcribed and analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets.Results: Total RNA yield from articular cartilage from primigravida rabbits was reduced to 65% of age-matched control values (P=0.0003); however the yield from multiparous animals was not significantly depressed. In both cases, DNA yields were not affected by pregnancy. There was a general tendency for depressed mRNA levels for most genes investigated in cartilage from pregnant animals. Articular cartilage from multiparous rabbits showed a significant decrease in mRNA levels for relevant molecules such as type II collagen, biglycan, collagenase and tissue inhibitors of metalloproteinases (TIMP)-1, as well as necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2 (COX-2). Transcripts for collagenase and lumican were significantly lower in cartilage from primigravida rabbits. Transforming growth factor β1 (TGF-β1) transcript levels were significantly decreased in both pregnant groups. In contrast, basic fibroblast growth factor (bFGF) and insulin-like growth factor-2 (IGF-2) mRNA levels were significantly decreased in cartilage from primigravida rabbits, whereas transcripts for these molecules were upregulated in the cartilage of multiparous rabbits.Conclusions: The present study demonstrates that regulation of RNA levels in articular cartilage during pregnancy is complex and is influenced by the parity and/or the skeletal maturity of the animals.

Proportions of fiber types in the external urethral sphincter of young nulliparous and old multiparous rabbits.

The proportions of fast and slow myosin molecules in specimen's of the external urethral sphincter (EUS) from 6 young nulliparous (6-month-old) and 6 old multiparous (2-year-old) rabbits were studied using myosin heavy chain electrophoresis. The percentages of fast and slow myosin molecules were 80.4 +/- 4.5% and 19.6 +/- 4.5% in the EUS from nulliparas, and 68.7 +/- 6.3% and 31.3 +/- 6.3% in the EUS from multiparas. The difference between the two groups was significant (P < 0.01). We suggest that a selective decrease in the volume of type 2 (fast) muscle fibers and/or conversion of type 2 to type 1 (slow) muscle fibers had taken place in the EUS of old multiparas. The proportional change in the constituent muscle fibers of the EUS with aging may play a role in human genuine stress incontinence.

The effect of teat anaesthesia on the milk-ejection reflex in the rabbit.

Section of the mammary nerves in goats and sheep or complete section of the spinal cord in the rabbit, rat, goat and cat (see review by Denamur, 1965) results in the suppression of suckling-induced milk ejection. In lactating women, Theobald (1959) failed to interrupt milk ejection by anaesthesia of the nipples produced by infiltration with 15 ml. of 1% lignocaine, but conditioning of oxytocin release to non-mammary stimuli may have occurred in these cases. In the present experiments the milk-ejection reflex was tested in six multiparous rabbits of mixed breed by the method of Cross & Harris (1952), in which litter weight gain during suckling is taken as an indicator of oxytocin release. The rabbits were separated from their litters on the day after parturition and daily suckling tests began at 10.00 hr. on the following day. A variety of treatments were applied before nursing. The teats were anaesthetized by

Changes in nest building behaviour in multiparous rabbits

This study deals with the effects of repeated litter production on one aspect of nest building behaviour, quality of nest. The results in the files of the Rabbit Genetics Laboratory, R. B. Jackson Memorial Laboratory, Bar Harbor, Maine,on over 800 rabbits of different races and sublines were utilised. From these extensive data, a group of 84 rabbits, selected on the basis of having produced four litters, were analysed. They were members of three different races and two sublines as follows: Race X, Race AcEp, Race III, and sublines IIIc and IIIr 2 . The hypothesis tested in the analysis was that there would be systematic improvement in performance with each of the four litters in the quality of nest production. Analyses of variance were made, which support the following conclusions: (1) a confirmation of the previous findings by Sawin & Crary (1953) of significant race differences; (2) significant improvement in quality of nest over the first four litters produced; (3) a good approximation of the functional relationship of nest rating to litters, as a linear one. The interpretation of this relationship is quite complex. Both learning and endocrine changes are possible hypotheses to be considered though no conclusion concerning the underlying mechanism is possible at this point. Further analyses and experimentation will be necessary to gain an understanding of this phenomenon.