Person: Lorenzo González, Pedro Luis
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First Name
Pedro Luis
Last Name
Lorenzo González
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Fisiología
Area
Fisiología
Identifiers
5 results
Search Results
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Item Physiological effects on rabbit sperm and reproductive response to recombinant rabbit beta nerve growth factor administered by intravaginal route in rabbit does(Theriogenology, 2020) Sánchez Rodríguez, Ana; Arias Álvarez, María; Millán Pastor, María Pilar; Lorenzo González, Pedro Luis; García García, Rosa María; García Rebollar, PilarBeta nerve growth factor (β-NGF) is present in the seminal plasma of some species, including rabbits, acting as an ovulation-inducing factor in camelids. Traditionally, GnRH analogues are used to induce ovulation by intramuscular route when artificial insemination (AI) is performed in rabbit does. A specific rabbit recombinant β-NGF (rrβ-NGF) produced in our laboratory was tested as an alternative method to conventional treatment with GnRH analogues to induce ovulation. In the present work, different concentrations (0, 20, 100 ng/mL and 1, 20 and 100 μg/mL) of rrβ-NGF were added to diluted semen to assess its effect on sperm traits (viability and motility parameters). rrβ-NG was used also, incorporated to the AI dose, to evaluate ovulation response (LH and progesterone plasma concentrations, ovulation rate (OR) and embryo implantation at Day 7) after intravaginal administration. A negative control group stimulated with an empty catheter, and a positive control group inseminated and intramuscularly treated as usual with GnRH were also set up. Results showed that seminal quality was influenced by rrβ-NGF depending on the concentration added, being the highest concentrations tested deleterious for semen. Whereas the highest OR was found in the positive control group (100%), concentrations of 20 ng/mL, 1 μg/mL and 20 μg/mL of rrβ-NGF triggered intermediate OR (30, 60 and 42.9%, respectively), and 100 ng/mL and 100 μg/mL had the lowest OR (20 and 14.3%, respectively). Although LH peak was not observed in the first 2 h after AI in the ovulated females from rrβ-NGF groups, plasma progesterone significantly increased at Day 7, except in those females treated with 20 and 100 μg/mL. Also, 98.4% of ovulated females were pregnant on Day 7. In conclusion, rrβ-NGF added to diluted semen affects seminal quality and provokes ovulation, the development of functional CL and conception by intravaginal route in rabbit does, depending on the concentration added.Item Improvements in the conception rate, milk composition and embryo quality of rabbit does after dietary enrichment with n-3 polyunsaturated fatty acids(Animal, 2018) Rodríguez, M.; García García, Rosa María; Arias Álvarez, María; Millán Pastor, María Pilar; Febrel, N.; Formoso-Rafferty Castilla, Nora; López-Tello, J.; Lorenzo González, Pedro Luis; Rebollar, P.G.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% (P<0.05) in terms of crown-rump length, biparietal and thoracic diameters, respectively, compared to control ones at first parturition. It is noteworthy that at the second insemination, critical point in rabbit, fertility rate of enriched group did not decline as sharply as in the control group (89.7% v. 76.6%, respectively; P=0.067), although ADG and littler weight were slightly lower at the second lactation after PUFA enrichment (P<0.05). Total PUFA and unsaturated index of milk of enriched does group were significantly elevated than in control one (33.3±0.02 v. 23.2±0.02 g/100 g and 1.20±0.00 v. 0.86±0.00, respectively; P<0.05). Finally, plasma progesterone, ovulation rate, fertility and embryo development at 3.5 days after the artificial insemination were similar between diets (P>0.05), 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.Item Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits(PLoSONE, 2019) Sánchez Rodríguez, Ana; Abad, Paloma; Arias Álvarez, María; Millán Pastor, María Pilar; García Rebollar, Pilar; Bautista Santa Cruz, José Manuel; Lorenzo González, Pedro Luis; García García, Rosa MaríaIn some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.Item Characterization of β-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between β-NGF and testosterone levels with seminal quality during sexual maturation(Theriogenology, 2019) Sánchez Rodríguez, Ana; Arias Álvarez, María; Timón, Patricia; Bautista Santa Cruz, José Manuel; García Rebollar, Pilar; Lorenzo González, Pedro Luis; García García, Rosa Maríaβ-Nerve Growth Factor (β-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of β-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. β-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine β-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate β-NGF concentration, and testosterone levels were also assessed in BP. The highest β-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual β-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of β-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, β-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. β-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and β-NGF.Item α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes(Reproduction, Fertility and Development, 2018) Arias Álvarez, María; García García, Rosa María; López-Tello, J.; Rebollar, P.G.; Gutiérrez-Adán, A.; Lorenzo González, Pedro LuisThe developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding a-tocopherol (0, 100, 200 and 400 mu M) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus-oocyte complexes (COCs) after IVM with 100 mu M alpha-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 mu M alpha-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 mu M alpha-tocopherol was included in the IVM medium, but remained low compared with in vivomatured oocytes. In conclusion, the addition of 100 mu M alpha-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.