Cristiano Boiti

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.

Changes in refractoriness of rabbit corpora lutea to a prostaglandin F2 alpha analogue, alfaprostol, during pseudopregnancy

The responsiveness of rabbit corpus luteum to 200 micrograms of the prostaglandin F2 alpha (PGF2 alpha) analogue, alfaprostol, between Days 3 and 9 of pseudopregnancy was assessed by evaluating the decline in plasma progesterone after treatment with PGF2 alpha in 81 New Zealand White (NZW) rabbits. On Days 3-5, functional luteolysis was not observed. On Days 6, 7, and 8 of pseudopregnancy, the number of rabbits responsive to PGF2 alpha, rose from 38% to 71% and 83%, respectively. In the other cases, the effect of the PGF2 alpha analogue was transient as CL recovered in the following 2 or 3 days. By contrast, on Day 9 luteolysis was effective and persistent in all the animals. In rabbits treated on Day 9, progesterone decreased gradually from 10.6 +/- 0.7 within the first 6 h, but fell to 3.6 +/- 1.5 ng/mL (p < 0.01) 12 h after PGF2 alpha and to 0.2 +/- 0.1 ng/mL (p < 0.01) 24 h later.

Ovulation induction in rabbit does: Current knowledge and perspectives

The profitability of rabbit farms has increased in recent years due primarily to improvements in the management of reproduction and genetic selection. This review summarizes the most important scientific papers relating to ovulation in rabbit does dealing in particular with: (a) studies from 1905 to the present day relating to ovulatory mechanisms in rabbit does; (b) research on the primary gonadotrophin-releasing hormone (GnRH), its analogues and their functions; and (c) descriptions of parenteral and intravaginal (iv.) treatments for induction of ovulation in does and their reported efficacies. The addition of GnRH analogues via the seminal dose (iv.) fulfils the need for a welfare-orientated method of inducing ovulation in rabbits. The structure, tissues, secretions, contractions, and innervations of the vagina in rabbits that can affect absorption profiles are reviewed in the context of recent reports of the achievement of high ovulation rates obtained by adding GnRH analogues directly to the seminal dose. This review demonstrates the possibility of ovulation induction in rabbits by the addition of GnRH synthetic analogues to the seminal doses and provides new perspectives for simplifying the AI technique.

In vitro effects of gonadotropin-releasing hormone (GnRH) on Leydig cells of adult alpaca (Lama pacos) testis: GnRH receptor immunolocalization, testosterone and prostaglandin synthesis, and cyclooxygenase activities.

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).

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.