Gabriele Brecchia

Doppler evaluation of maternal and fetal vessels during normal gestation in rabbits

The aim of this work is to evaluate the haemodynamic characteristics of maternal and foetal vessels during normal pregnancy in queens, using colour Doppler and pulsed wave Doppler ultrasonography, in order to obtain information about maternal and foetal circulation. The blood waveforms of the uteroplacental arteries, aorta, caudal cava vein and umbilical cord of the fetuses were recorded weekly in seven healthy pregnant queens. Also, the measurements of peak systolic, end diastolic velocities, resistance and pulsatility indices were carried out. Uteroplacental blood flow was biphasic while the ones of the umbilical artery and aorta were first systolic and then diastolic. The caudal cava vein showed a typical waveform of venous vessels. During gestation the EDV and PSV of foetal vessels increased (alpha<0.05) while the PI and RI of all vessels examined decreased (alpha<0.05) except for the IP of the aorta. The Doppler ultrasonography, also in queens, can be used to evaluate the characteristics of maternal and foetal vessel flow and their progressive changes during pregnancy. This study can be considered the basis for further contribution in diagnosing and monitoring high-risk pregnancies in Veterinary Medicine.

Reproductive rhythm and litter weaning age as they affect rabbit doe performance and body energy balance

Abstract One hundred and twenty multiparous does were synchronized to give birth the same day (initial kindling). The trial lasted until the successive (final) kindling. Immediately following initial kindling, 22 does were selected for comparative slaughter. The remaining does were assigned to three reproductive rhythms and mated 2 (R2), 11 (R11) or 26 (R26) days post partum. Within each rhythm, the does were further divided into two groups with litters weaned at 21 (W21) or 25 (W25) days of age. A total of 54 does were pregnant and were slaughtered soon after final kindling. By increasing the kindling-to-mating interval from 2 to 26 days, total milk production was increased (5590 to 6065 g for R2 and R26, respectively; P P P P P P P P P P P P P

Intraluteal regulation of prostaglandin F2α-induced prostaglandin biosynthesis in pseudopregnant rabbits

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

Metabolic adaptation and hormonal regulation in young rabbit does during long-term caloric restriction and subsequent compensatory growth

An experiment was performed to assess the metabolic adaptation and hormonal regulation in young female rabbits during long-term food restriction and subsequent compensatory growth during rearing. Feeding level was either ad libitum (AL, no. = 52) or restricted (R, no. = 52). From 6 to 12 weeks of age, food intake of R was kept at a constant level. This resulted in an increase in relative restriction as compared with AL to 0.54of AL intake at 12 weeks of age (restriction period). Thereafter food intake gradually increased to 0.95 of AL at 17 weeks of age (recovery period). During the last 5 days before insemination at 17.5 weeks of age, all animals were fed to appetite. Blood samples were taken weekly from 6 to 17 weeks of age from 11 animals in each group. Growth rate of R was reduced during the restricted period (29 (s.d. 2) v. 44 (s.d. 5) g/day for R and AL, respectively; P <0.05), but was higher in the recovery period (30 (s.d. 3) v. 27 (s.d. 4) g/day, respectively; P <0.05). At first insemination, AL rabbits were heavier than R (4202 (s.d. 388) v. 3798 (s.d. 220) g, respectively; P <0.001). During the restricted period, plasma glucose was constantly lower (P <0.05) in R. Insulin levels paralleled those of glucose, being lower (P <0.05) in R than in AL. Restriction reduced CP <0.05) circulating corticosterone and tri-iodothyronine (T3) levels in R. Leptin, non-esterified fatty acids, and plasma urea nitrogen levels were similar for AL and R during food restriction, whereas triglycerides were similar until 10 weeks of age, after which the levels were lower in R. During the recovery period, the food intake of the R but not AL rabbits increased. Insulin was the only hormone in R rabbits that had returned to levels found in AL rabbits by the 2nd week of the recovery period. Glucose, T3, and corticosterone levels returned to levels found in AL rabbits between 3 to 4 weeks after refeeding. Non-esterified fatty acids, triglycerides, and leptin were higher (P <0.05) in AL rabbits from 13 weeks of age onwards. The pattern of changes in the endocrine status during food restriction and compensatory growth in rabbits do conform with those from other species, although some specific changes may vary depending on the severity of food restriction and its duration.

Evidence for a Luteotropic Role of Peroxisome Proliferator-Activated Receptor Gamma: Expression and In Vitro Effects on Enzymatic and Hormonal Activities in Corpora Lutea of Pseudopregnant Rabbits

ABSTRACT The expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and its role in corpora lutea (CL) function were studied in pseudopregnant rabbits. Corpora lutea were collected at an early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. Immunohistochemistry found evidence for the presence of PPARgamma in the perinuclear cytoplasm and nucleus of all the luteal cells; immunoreactivity decreased from the early to the late stage, with immunonegativity of the nuclei of late stage CL. PPARgamma mRNA transcript was expressed in all the luteal stages with the lowest level in the late stage. In CL cultured in vitro, the PPARgamma agonist (15-deoxy delta12,14 prostaglandin J2 [15d-PGJ2], 200 nM) increased and the antagonist (T0070907, 50 nM) decreased progesterone secretion at early and midluteal stages, whereas 15d-PGJ2 reduced and T0070907 increased PGF2alpha at the same stages. Prostaglandin-endoperoxide synthase 2 (PTGS2) activity was reduced by 15d-PGJ2 and increased by T0070907 in CL of early and midluteal stages. Conversely, 15d-PGJ2 increased and T0070907 reduced 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in early and midluteal stage CL. PGE2 in vitro secretion as well as PTGS1 and 20alpha-HSD enzymatic activities were not affected by 15d-PGJ2 and T0070907 in any CL types. These results indicate that PPARgamma plays a luteotropic role in pseudopregnant rabbits, through PTGS2 down-regulation and 3beta-HSD up-regulation, with a consequent PGF2alpha decrease and progesterone increase.

Expression of luteal estrogen receptor, interleukin-1, and apoptosis-associated genes after PGF2α administration in rabbits at different stages of pseudopregnancy

The dynamic expression for estrogen receptor subtype-1 (ESR1), interleukin-1beta (IL1B), and apoptosis-associated genes, as well as nitric oxide synthase activity, were examined in corpora lutea (CL) of rabbits after prostaglandin F(2alpha) (PGF(2alpha)) administration on either day 4 or day 9 of pseudopregnancy. By reverse transcriptase polymerase chain reaction, the steady-state level of ESR1 transcript was lower (P < 0.01) and that of anti-apoptotic B-cell CLL/lymphoma 2 (BCL2) -like 1 (BCL2L1) was greater in day 4 (P < 0.01) than in day 9 CL. Western blot analysis revealed that BCL2-associated X protein (BAX) abundance was greater in day 4 (P < 0.01) than in day 9 CL, whereas BCL2L1 protein was undetectable at both luteal stages. After PGF(2alpha), ESR1 transcript decreased (P < 0.01) in day 9 CL, whereas IL1B mRNA showed a transitory increase (P < 0.01) at both stages. The pro-apoptotic tumor protein p53 (TP53) gene had diminished (P < 0.01) on day 4 and on day 9 after a transitory increase (P < 0.01), whereas the BAX/BCL2L1 expression ratio increased (P < 0.01) in day 9 CL 24 h after treatment. Following PGF(2alpha), TP53 protein increased (P < 0.01) at both luteal stages, and BAX decreased (P < 0.01) in day 4 CL but increased (P < 0.01) 24 h later in day 9 CL; BCL2L1 became detectable 6 h later in day 4 CL. Nitric oxide synthase activity temporarily increased (P < 0.01) following PGF(2alpha). These findings suggest that PGF(2alpha) regulates luteolysis by ESR1 mRNA down-regulation and modulation of pro- and anti-apoptotic pathways in CL that have acquired a luteolytic capacity.

Short- and long-term effects of lipopolysaccharide-induced inflammation on rabbit sperm quality

Infections and resulting inflammation are widely known to cause transient or permanent male infertility. The objectives of this study were (1) to provide a suitable animal model of a sub-acute inflammatory state by intraperitoneally inoculating bacterial lipopolysaccharides (LPS) and (2) to define the short- and long-term effects of this state on the sperm quality of rabbit bucks. Two series of experiments were performed to accomplish these objectives. In experiment 1, 15 healthy New Zealand White rabbit bucks were divided into five homogeneous groups, receiving 25, 50, 100 and 150 microg/kg body weight (b.w.) of E. coli LPS dissolved in 2ml of sterile saline or only saline (control), respectively. White blood cells (WBC), rectal temperature, feed intake and mating ability were observed for 3 consecutive days following inoculation. Inoculation of 50 microg/kg b.w. produces a reversible inflammation-like state that lasts for about 3 days, with minimal distress to the animals, and therefore it was used in our experiment. The major symptoms were fever and anorexia. Changes in WBC count and a moderate reduction in reproductive activity also occurred. In experiment 2, two groups of five rabbit bucks each were treated with 50 microg/kg b.w. E. coli LPS diluted in 2ml of saline or only saline (controls), respectively. Semen samples were collected weekly up to 56 days after inoculation and the changes in semen characteristics were examined. During the first 3 days, semen volume and concentration decreased in both experimental groups, probably due to the high collection frequency. Sperm membrane integrity and the number of necrotic sperm were seriously affected 30 days after the LPS challenge, reaching a maximum at the end of the spermatogenic cycle (56 days). These results suggest that a sub-acute inflammation may cause infertility by compromising sperm membrane integrity which decreases a month after LPS-treatment. In addition, the rabbit could be a useful LPS animal model for further study of the effects of inflammation and the underlying mechanisms on sperm characteristics.

Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24  h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE₂ at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP₃, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP₃, DAG, PKC, COX-2, and NOS.

Effects of induced endometritis on the life-span of corpora lutea in pseudopregnant rabbits and incidence of spontaneous uterine infections related to fertility of breeding does.

A significant percentage of rabbit does fail to become pregnant after AI. We hypothesized that uterine infections induced by the insemination procedure are related to delayed luteolysis and high progesterone concentrations noted to present at the time of AI. The rabbits, randomly assigned to 4 groups (3 animals/group), were given 0.8 microgram GnRH analogue (Day 0) just prior to infusing the uterus with sterile extender (control group) or with extender inoculated with 0.5, 1, and 2 x 10(6) Pasteurella multocida (treated groups). The effects of treatments on functional life-span of CL were assessed by evaluating plasma progesterone from Day 0 to Day 23 of pseudopregnancy. In treated rabbits, the progesterone profiles closely overlapped those found in controls until approximately Day 14. Thereafter, they varied greatly between animals, but luteolysis was delayed by at least 5-6 d and developed less rapidly than in controls. On Day 21, progesterone concentrations were higher than normal in 4 treated does. In a field survey, vaginal swabs were collected at the time of the second AI from 114 non-pregnant rabbits and those positive to bacteriological culture, were killed humanely 16 d later to collect uterine swabs. Positive uterine swabs were found only in 19 of the 34 does having a positive vaginal swabs and all of them were not pregnant. The most frequent pathogen isolated was S. aureus (50%), followed by E. coli (37.5%) and P. multocida (12.5%). We demonstrated that uterine infection increases the life-span of CL in non-pregnant does and that infections of the genital tract system are quite common among does on breeding farms, probably related to using AI.

Aglepristone (RU534) administration to non-pregnant bitches in the mid-luteal phase induces early luteal regression

The effect of the antiprogestagen aglepristone (10 mg/kg bw), administered at days 29 and 30 following the estimated day of LH surge (day 0), on corpora lutea (CL) function was examined during the diestrus phase of non-pregnant bitches. Aglepristone shortened (P < 0.01) the luteal phase and complete luteolysis (progesterone <2 ng/mL) was observed at days 40.8 +/- 3.5 and 71.5 +/- 4.6 (means +/- SD; n = 9/group) in treated and control bitches, respectively. Peripheral estradiol-17beta concentrations declined from 91.5 +/- 14.3 pg/mL at day 9 to 50 pg/mL at day 18, remaining at approximately the same levels thereafter in both treated and control bitches. Intraluteal in vitro synthesis of progesterone and estradiol-17beta released by CL explanted at day 38 from control bitches (511.9 +/- 285.6 and 40.7 +/- 17.2 pg/mg protein, respectively) did not differ from that of treated. From day 38, intraovarian hemodynamic variables (arterial blood flow, systolic peak, and end-diastolic velocities), monitored by color-coded and pulsed Doppler, decreased more steeply (P < 0.01) in aglepristone-treated (n = 4) than in control (n = 4) bitches, whereas the resistance index increased (P < 0.01) in treated animals. All the blood flow parameters were undetectable at 60 +/- 3.6 and 68 +/- 2.0 days (medians +/- SD) after LH peak in treated and control bitches, respectively. In conclusion, aglepristone administration to dogs during the mid-luteal phase markedly accelerates the luteolytic process which is accompanied by a parallel decline in ovarian blood flow supply with a shift from approximately 8 to 10 days.