P. Millán

Influence of metabolic status on oocyte quality and follicular characteristics at different postpartum periods in primiparous rabbit does

Low reproductive performance of high-yield primiparous animals is closely associated with the metabolic stress caused by a simultaneous gestation and lactation. The aim of this work was (1) to analyze body composition and metabolic environment at three time points along lactation (at parturition time; in the lactation period [Day 11 postpartum]; and in the postweaning period [Day 32 postpartum]) of primiparous rabbit does (Oryctolagus cuniculus) and (2) to investigate the ovarian status at insemination time and the possible link with metabolic environment and with their reproductive performance. To this end, does were either submitted to a semi-intensive reproductive rhythm (Group S, inseminated on Day 11 postpartum) or an extensive rhythm (Group E, inseminated on Day 32 postpartum). Body energy (P<0.05) and protein content (P<0.001) as well as serum leptin (P<0.05) and protein concentrations (P<0.001) increased significantly along the postpartum period. At parturition, body lipid content was significantly lower and serum nonesterified fatty acids concentrations were significantly higher than that on Days 11 postpartum and 32 postpartum. Concerning ovarian status at insemination time, no significant differences were found in mean follicular stages, serum estradiol, progesterone, and prolactin (PRL) concentrations or in prolactin receptor (PRL-R) immunostaining. However, follicles in Group S showed a significantly higher apoptosis index than that of Group E (P<0.001). The nuclear and cytoplasmic oocyte maturation rates of Group S were also significantly lower than that in Group E. In addition, conception rate and prolificacy were improved in Group E (P<0.001 and P<0.05, respectively). In conclusion, in the early postpartum period, metabolic status seems to impact negatively on ovarian follicle and oocyte quality leading to a poor reproductive outcome in primiparous rabbit does.

Follicular, Oocyte and Embryo Features Related to Metabolic Status in Primiparous Lactating does Fed with High-Fibre Rearing Diets

Fertility of primiparous lactating does in the early postpartum (pp) period is very low mainly due to pronounced deficient energy intake, influencing oocyte and embryo developmental competence. The hypothesis used in this work was that high-lignin fibre diet supplied during the rearing period could increase feed intake and, consequently, improve the reproductive physiology and metabolic status of primiparous does in the early pp period. Diets with high-lignin [HL: 15.8% dry matter (DM)] or standard-lignin content (SL: 4.9% DM) were supplied until parturition time. No diet effects in serum oestradiol, progesterone concentrations and follicle categories were found in the histological study. Metaphase II rate of in vitro-matured oocytes was significantly higher in the SL vs the HL group (p < 0.001). Cytoplasmically degenerated oocytes (in terms of abnormal distribution of cortical granules) and follicular atresia rate were significantly lower in the SL group than in the HL group (p < 0.05 and p < 0.005 respectively). In addition, HL-fed does showed lower number of viable embryos and higher rate of retarded in vivo-recovered embryos compared with the SL group (p < 0.05). Neither in vitro embryo development of viable embryos nor conception rate was significantly different between groups. Feed intake increased during the first pregnancy in the HL group (p < 0.05), but not during early lactation. Serum protein, non-esterified fatty acid and leptin concentrations, as well as estimated body composition were similar in does fed with both diets. In conclusion, the enhancement of reproductive management by using highly lignified products in rearing diets does not seem to report physiological reproductive benefits affecting oocyte maturation rate and embryo viability in primiparous lactating does.

Expression of the cannabinoid receptor type 1 in the pituitary of rabbits and its role in the control of LH secretion

The aim of this study was to elucidate the possible direct regulatory role of the endocannabinoids in the modulation of LH secretion in rabbits, a reflex ovulator species. The cannabinoid receptor type 1 (CB1) was characterized by RT-PCR techniques in the anterior pituitary of intact and ovariectomized does treated with GnRH and primed with estrogen and CB1 antagonist, rimonabant. Cannabinoid receptor type 1 immune reaction was evidenced by immunohistochemistry in the cytoplasm of approximately 10% of the pituitary cells with a density of 8.5 ± 1.9 (per 0.01 mm(2)), both periodic acid-Schiff positive (30%) and negative (70%). All CB1-immunoreactive cells were also immune reactive for estrogen receptor type 1. Ovariectomy, either alone or combined with estrogen priming, did not modify the relative abundances of pituitary CB1 mRNA, but decreased (P < 0.01) the expression of estrogen receptor type 1 mRNA. Treatment with CB1 antagonist (rimonabant) inhibited (P < 0.01) LH secretory capacity by the pituitary after GnRH injection, and estrogen priming had no effect. The present findings indicate that the endocannabinoid system is a potential candidate for the regulation of the hypothalamic-pituitary-ovarian axis in reflex ovulatory species.

Isoflavones and their effects on the onset of puberty in male Wistar rats.

This study was performed to determine how two of the most important isoflavones, genistein and daidzein, affect the gonadal axis in male prepuberal rats. One hundred and seventy‐five prepuberal male Wistar rats were allocated into seven groups: one control group and six experimental groups that were orally administered a high or low dose of genistein, daidzein or a mixture of both. Testosterone determination was assayed by EIA. The testes and body weights were measured, and the histology of the epididymis with the sperm content and epididymal sperm count were evaluated. In the control group, we observed an increase in the serum testosterone levels (>2.5 ng ml−1) at the third week (52 days), which corresponded to the onset of puberty in these rats. The same increase in serum testosterone levels was observed at the fourth week in rats that received low doses of isoflavones; therefore, we concluded that the onset of puberty was delayed. At high doses, there was no significant increase in testosterone levels, which could be related to the fact that these male rats did not reach puberty. These findings were supported by the results obtained from the analysis of the epididymal content as well as the testes/body weight ratio.

The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty on Male Wistar Rats

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.

Body reserves and ovarian performance in primiparous lactating rabbit does submitted to early weaning as a strategy to decrease energy deficit

The aim of this work was to study the effect on body composition, serum metabolic parameters and ovarian status of early weaning at 25 Days post-partum (dpp) as a strategy to decrease energy deficit of primiparous lactating rabbit does prior to insemination at 32 dpp following an extensive rhythm. A total of 34 primiparous lactating rabbit does were used and distributed in three groups: 10 lactating does euthanized at 25 dpp (group L25), 13 does weaned at 25 dpp and euthanized at 32 dpp (group NL32), and 11 non weaned lactating does euthanized at 32 dpp (group L32). No significant differences were observed in live body weight, ovary weight, serum NEFA and total protein concentration among groups. Although NL32 does had a low feed intake (122+/-23.5g/Day; P<0.001), their estimated lipids (16.9+/-1.09%, P<0.008), protein (19.7+/-0.07%, P<0.0001), and energy (1147+/-42.7MJ/kg, P<0.006) body contents were higher and their serum glucose concentrations (158+/-24.5mg/dl, P<0.04) were lower compared to L25 does (11.9+/-1.3%, 18.5+/-0.08%, 942+/-51.3MJ/kg and 212+/-27.9mg/dl) and L32 does (13.4+/-1.03%, 18.5+/-0.1%, 993+/-40.4MJ/kg and 259+/-29.5mg/dl), respectively. In the ovarian surface of L25 does a lower number of follicles > or =1mm was observed compared to NL32 and L32 groups (12.7+/-1.5 vs. 18.0+/-1.45 and 17.6 +/-1.67; P<0.05). Follicular population in the histological ovarian sections and immunolocalization of prolactin receptor were similar between groups. In group L25, both nuclear maturation of oocytes in terms of Metaphase II rate (67.0 vs. 79.7 and 78.3%; P<0.05) and cytoplasmic maturation measured by percentage of cortical granules (CG) totally or partially migrated in oocytes were significantly lower than in groups NL32 and L32 (16.0 vs. 38.3 and 60.0%; P<0.05). Consequently, a higher rate of oocytes with non-migrated CGs was found in group L25 than in groups NL32 and L32 (76.0 vs. 46.8 and 33.3%; P<0.05). In conclusion, even though early weaning at 25 dpp seemed to improve body energy stores of primiparous does, this fact was not well reflected on the ovarian status at 32 dpp, which was similar regardless of weaning time and it could be performed later.

Improvements in the conception rate, milk composition and embryo quality of rabbit does after dietary enrichment with n-3 polyunsaturated fatty acids

This work attempts to confirm the effect of an enriched diet with n-3 polyunsaturated fatty acids (PUFA) trying to mitigate the reproductive performances issues such as low conception rate of primiparous rabbits. A total of 127 does were fed ad libitum throughout their two first cycles with two diets with different fat sources: mixed fat in the control and salmon oil in the enriched one, with 3.19 g/100 g (n=63 does) and 28.77 g/100 g (n=64 does) of n-3 of the total fatty acid, respectively. Feed intake was similar between groups (P>0.05). Plasma progesterone concentration was higher in the enriched females than in control ones at 7 (30.9±2.18 v. 23.9±2.30 ng/ml, respectively; P=0.029) and 14 (38.7±2.18 v. 28.2±2.30 ng/ml, respectively; P=0.001) days of first gestation. Considering both cycles, reproductive parameters of mothers (fertility, duration of gestation and prolificacy) and litter parameters (weight at parturition and weaning, mortality and average daily gain (ADG) of kits during lactation) were similar in both groups. However, individual measurements of neonates of enriched group improved 5.87%, 7.10% and 18.01% (P0.05), but embryo apoptosis rate was higher in control group than in enriched one (31.1±4.56% v. 17.1±3.87%, respectively; P<0.05). In conclusion, dietary PUFA enrichment from the rearing and throughout two productive cycles improved plasma progesterone during pregnancy, fertility, milk fatty acid profile and neonates development of primiparous supporting the beneficial effect of n-3 PUFA supplementation in rabbit does.

β-nerve growth factor identification in male rabbit genital tract and seminal plasma and its role in ovulation induction in rabbit does

Abstract The neurotrophin β-nerve growth factor (NGF) has been described as an important mediator of seminal plasma-induced ovulation in reflexive ovulators like camelids, but this action has not been evidenced in rabbits. In this study, the mRNA expression of NGF was analysed in rabbit male organs by PCR and in a purified basic fraction of seminal plasma by liquid chromatography mass spectrometry. Besides, the ovulatory response [ovulation rate (OR), corpora lutea number, LH peak and progesterone profile] of 27 rabbit does was recorded after injection with murine NGF, gonadoreline (GnRH) or saline solution (SS). An empty catheter was introduced through the vagina (n = 4 animals per group) to simulate the nervous/mechanical stimulus of coitus. Molecular studies confirmed the expression of β-NGF mRNA in the prostate, seminal gland and testicles of male adult rabbits, and the presence of the protein in seminal plasma. For the in vivo experiment, all GnRH-group does exhibited a significantly higher OR (100%) compared to females treated with NGF (30%) and SS (25%), who showed significantly lower LH and progesterone concentrations at 120 min. However, in the non-vaginally stimulated animals, only the gonadoreline group ovulated properly, since no ovulation was recorded in the SS group, and only 17% of the NGF does ovulated; this was associated to a tendency to a higher rate of anovulatory haemorrhagic follicles. mRNA expression of NGF and its presence in seminal plasma evidence a role in rabbit reproduction. Nevertheless, murine NGF triggered some stimulatory effect in the females but not enough to elicit a significant ovulatory response.

Induction of ovulation in rabbits by adding Lecirelin to the seminal dose: In vitro and in vivo effects of different excipients

This study evaluates the effect of Lecirelin (Dalmarelin(®), Fatro, Italy) diluted in different excipients (benzilic alcohol, benzoic acid and paraben) added to a seminal dose on LH concentrations, progesterone concentrations and ovarian status in rabbits. The in vitro effect on spermatozoa was also tested. A total of 100 multiparous female rabbits were divided into 5 groups, which at the moment of AI, received 0.2 mL (5 μg/dose) intramuscular (im) inoculation of Lecirelin (control) or the same Lecirelin dose administered intravaginally (iv) with the seminal dose alone (Lecirelin group) or with benzilic alcohol (Lecirelin BA group), benzoic acid (Lecirelin BAc group) or parabens (Lecirelin PA group) as an excipient. After 7 days, 10 rabbits per group were euthanized to analyze their ovarian status. In the control group, a high LH peak was detected 30 min post AI, while in the iv groups a slight increase in LH occurred after 120 min. The ovulation and fertility rate was similar in control and Lecirelin groups, while the lowest fertility rate was detected in the Lecirelin BA group. In a second experiment, the semen samples collected from male rabbits were diluted in TALP (control) or mixed with the 5 μg of Lecirelin solutions used in the first experiment. The highest percentage of capacitated sperm (68.3%) was recorded in the Lecirelin PA. The lowest percentages were observed in the Lecirelin BA and BAc groups. In conclusion, the iv administration of Lecirelin represents an alternative method for simplifying rabbit insemination procedures.

New insights on a NGF-mediated pathway to induce ovulation in rabbits (Oryctolagus cuniculus)

Abstract To investigate the ovulatory mechanisms triggered by raw semen (RS) in rabbits, we examined the expression of nerve growth factor (NGF)—a supposed ovulation-inducing factor (OIF)—and cognate receptors in anterior pituitary, ovary, and cervix as well as plasma NGF and luteinizing hormone (LH) concentrations. Six does/group were sham-inseminated with sterile saline (PBS), naturally mated (NM), inseminated with RS alone or after lumbar anesthesia (ARS), or treatment with COX inhibitors (CIRS). Immunohistochemistry revealed positive signals for NGF and receptors in all tissues. RT-PCR confirmed the presence of the target transcripts in the same tissues, except NTRK1 in the cervix. Circulating NGF concentrations rose 3- to 6-fold (P < 0.01) 15 min after semen deposition into the genital tract of NM, RS, and ARS rabbits and remained sustained thereafter. Circulating NGF was 4-fold lower (P < 0.01) in CIRS than in RS does indicating that NGF is mainly synthesized by the uterus. A concomitant rise of LH and NGF concentrations was found in 83.3%, 50.0%, and 16.7% of NM, RS, and CIRS does, respectively, but not in ARS (despite high NGF circulating levels). Seminal plasma NGF concentration was 151.9 ± 9.25 µg/mL. The ovulatory responses were 0%, 83.3%, 66.7%, 16.7%, and 0% in PBS, NM, RS, ARS, and CIRS groups, respectively. Present data confirm that, although RS may induce ovulation via endocrine mechanisms through binding to NGF receptors in the ovary, a novel OIF-mediated neural mechanism facilitates ovulation in rabbits. Summary Sentence Raw semen induces ovulation in rabbits via an endocrine- and a nervous-mediated pathway by which NGF, mainly synthesized in the uterus, acts on the ovary and on uterine/cervix afferent neurons projecting LH surge hypothalamic centers, respectively.