Ghrelin antagonist overrides the mRNA expression of NPY in hypothalamus in feed restricted ewes

A negative energy balance (NEB) is detrimental to reproduction in animals. A suggested link between NEB and reproductive failure is the gastrointestinal hormone ghrelin, because of the association between ghrelin and the hypothalamo-pituitary-gonadal axis. The [D-Lys3]-Growth Hormone Releasing Peptide-6 ([D-Lys3]-GHRP-6) is a ghrelin antagonist that acts on ghrelin receptors (GHS-R1). The objective of this study was to evaluate the effect of [D-Lys3]-GHRP-6 on reproduction variables in feed restricted ewes. Two experiments were conducted. Experiment I was conducted for 30 days; and Experiment II for 13 days. In both experiments the ewes (n = 18) were randomly assigned to: Control (CO): fed to meet maintenance requirements; Feed restriction (FR): 80% of maintenance restriction; or Ghrelin antagonist (GA): feed restricted and daily subcutaneous of 7.5μg/kg of [D-Lys3]-GHRP-6. Plasma was collected to measure hormones and metabolite concentration. In Experiment II, the hypothalamus and ovaries were collected on day 13. In both Experiments, sheep allocated to the FR and GA treatments decreased their body weight compared with sheep in the CO group (P 0.10). Experiment I: Plasma ghrelin concentration was greater (P < 0.01) in FR and GA compared with CO ewes. Plasma non-esterified fatty acids concentration was greater (P < 0.01) in GA and FR than CO. Experiment II: Kisspeptin1-Receptor (Kiss1-R) mRNA expression was greater in FR (P < 0.01) and tended to be greater in GA (P = 0.10) compared with CO ewes. The neuro peptide-Y (NPY) mRNA expression was greater (P = 0.03) in FR than CO; and tended to be greater (P = 0.06) compared with GA ewes. Growth hormone releasing hormone (GhRH) mRNA expression was greater in GA (P = 0.04) and tended to be greater in FR (P = 0.07) compared with CO ewes. Feed restriction increased GhRH, NPY, and Kiss-R mRNA expression in hypothalamus without affecting reproductive variables.Ghrelin antagonist may prevent an increase inNPY expression in ewes.Fil: Carranza Martin, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Ohio State University; Estados UnidosFil: Parker, Anthony J.. Ohio State University; Estados UnidosFil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Relling, Alejandro Enrique. Ohio State University; Estados Unido

Expression of vascular endothelial growth factor in normal and tumoral liver mouse cells

In previous studies have been reported a controversy between the reactivity of mono and polyclonal antibodies in different cells populations of the mouse. In this study we focus on the immunoreactivity of the monoclonal antibody VEGF (C- 1) in order to analyze its expression in two cell populations of regenerating mouse liver (hepatocytes and endothelial cells) after partial hepatectomy, and two transplanted hepatocarcinomas (ES2 and SS1K). C3H/S male mice were divided into two groups and kept under standard conditions for circadian periodicity analysis, one group were transplanted with the tumors into the subcutaneous tissue of the animal flank and the other group were subjected to a partial hepatectomy (70 %). The animals were killed by decapitation and exsanguination. Samples of regeneration liver, ES2 and SS1K tumors were processed for histology and immunostained with VEGF (C-1) mouse monoclonal IgG2a antibody. The results showed strongly immunopositivity reaction for VEGF either in hepatocytes or endothelial cells of mouse regenerating liver. We also demonstrated positive immunoreaction in both malignant tumors ES2 and SS1K endothelial and parenchymal cells. We conclude that the use of VEGF (C-1) mouse monoclonal IgG2a antibody can be applied successfully in experimental design leading to study processes involved in mice’s angiogenesi

Prepartum fatty acid supplementation in sheep : III. Effect of eicosapentaenoic acid and docosahexaenoic acid during finishing on performance, hypothalamus gene expression, and muscle fatty acids composition in lambs

The objectives of this study were to evaluate the effect of feeding an enriched diet with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to finishing lambs born from ewes supplemented either with or without EPA and DHA during late gestation on productive performance, muscle fatty acid (FA), and hypothalamus mRNA concentration of metabolic genes and hormone receptors. Lambs born from dams fed during the last 50 d of gestation either with a control diet containing 0.39% Ca salts of palmitic fatty acid distillate (C) or Ca salts enriched with EPA and DHA (PFA) were used. After weaning lambs (n = 70) were blocked by weight (BW) and used in a 2 × 2 factorial into 2 finishing diets containing 1.5% of C or PFA. The 2 factors were the ewe diet and the finishing diet. Lambs (37.9 ± 0.4 kg) were weighed and blood sampled for glucose and NEFA measurements at days 1, 14, 28, and 42. Dry matter intake (DMI) was measured daily. At day 43, 14 females and 14 males were slaughtered, and hot carcass weight, body wall thickness, rib eye area, and FA composition of Longissumus thoracis muscle were evaluated. Female hypothalamuses were obtained and mRNA concentration of hormone receptors, neuropeptides, and their receptors was measured. Lambs born from PFA dams were heavier (P 0.1). Lambs born from PFA dams had a greater concentration of C22:0 (P < 0.03). Lambs fed C had higher concentrations of C18:1c15 (P < 0.01), C17:0 (P < 0.09), C18:0 (P < 0.09), and n6/n3 (P < 0.01). Lambs fed PFA had greater concentration (P < 0.05) of C16:1, C22:1, C20:5, C22:5, C22:6, total n3 FA, and total EPA and DHA. There was a significant dam × finishing diet interaction (P ≤ 0.08) on mRNA concentration for MCR3, CCK-R, Cort-R, and CART. Lambs, which had the same treatment as their dams, showed lower overall mRNA concentration than those with different treatments between them and their dams. Lambs born from PFA ewes had lower concentration of MCR4 mRNA (P = 0.09) than C. Agouti-related peptides mRNA concentration was lower in lambs fed PFA (P = 0.06) than C. In conclusion, changes on lamb performance, muscle fatty acid composition, and metabolic neuropeptides depend not only on the lamb diet, but also on the dam diet during late gestation.Instituto de Genética Veterinari

Prepartum fatty acid supplementation in sheep : III. Effect of eicosapentaenoic acid and docosahexaenoic acid during finishing on performance, hypothalamus gene expression, and muscle fatty acids composition in lambs

The objectives of this study were to evaluate the effect of feeding an enriched diet with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to finishing lambs born from ewes supplemented either with or without EPA and DHA during late gestation on productive performance, muscle fatty acid (FA), and hypothalamus mRNA concentration of metabolic genes and hormone receptors. Lambs born from dams fed during the last 50 d of gestation either with a control diet containing 0.39% Ca salts of palmitic fatty acid distillate (C) or Ca salts enriched with EPA and DHA (PFA) were used. After weaning lambs (n = 70) were blocked by weight (BW) and used in a 2 × 2 factorial into 2 finishing diets containing 1.5% of C or PFA. The 2 factors were the ewe diet and the finishing diet. Lambs (37.9 ± 0.4 kg) were weighed and blood sampled for glucose and NEFA measurements at days 1, 14, 28, and 42. Dry matter intake (DMI) was measured daily. At day 43, 14 females and 14 males were slaughtered, and hot carcass weight, body wall thickness, rib eye area, and FA composition of Longissumus thoracis muscle were evaluated. Female hypothalamuses were obtained and mRNA concentration of hormone receptors, neuropeptides, and their receptors was measured. Lambs born from PFA dams were heavier (P 0.1). Lambs born from PFA dams had a greater concentration of C22:0 (P < 0.03). Lambs fed C had higher concentrations of C18:1c15 (P < 0.01), C17:0 (P < 0.09), C18:0 (P < 0.09), and n6/n3 (P < 0.01). Lambs fed PFA had greater concentration (P < 0.05) of C16:1, C22:1, C20:5, C22:5, C22:6, total n3 FA, and total EPA and DHA. There was a significant dam × finishing diet interaction (P ≤ 0.08) on mRNA concentration for MCR3, CCK-R, Cort-R, and CART. Lambs, which had the same treatment as their dams, showed lower overall mRNA concentration than those with different treatments between them and their dams. Lambs born from PFA ewes had lower concentration of MCR4 mRNA (P = 0.09) than C. Agouti-related peptides mRNA concentration was lower in lambs fed PFA (P = 0.06) than C. In conclusion, changes on lamb performance, muscle fatty acid composition, and metabolic neuropeptides depend not only on the lamb diet, but also on the dam diet during late gestation.Instituto de Genética Veterinari

Hyaluronan and Hyaluronidase, which is better for embryo development?

Our aim was to examine size-specific effects of Hyaluronan (HA) on preimplantation embryo development. We investigated the effects of Hyalovet (HA, 500–750 kDa; the size produced by HA synthase-3, which is abundant in the oviduct), or HA treated with Hyaluronidase-2 (Hyal2; also expressed in the oviduct that breaks down HA into 20 kDa fragments). In experiment 1 (in vivo), oviducts of synchronized and superovulated ewes (n = 20) were surgically exposed on Day 2 post-mating, ligated, and infused with either Hyalovet, Hyalovet + Hyal2, Hyal2, or PBS (control). Ewes were killed 5 days later for recovery of embryos and oviductal epithelial cells (OEC). Blastocyst rates were significantly higher in Hyal2 and Hyalovet + Hyal2 oviducts. Hyaluronidase-2 infusion resulted in higher blastocyst cell numbers and hatching rates. This was associated with increased HSP70 expression in OEC. In contrast, Hyalovet resulted in the lowest development to blastocyst stage and lowest hatching rates, and decreased IGF2 and IGFBP2 expression in OEC. IGF1 and IL1α expression were not affected. In experiment 2, to rule out indirect effects of oviductal factors, ovine embryos were produced and cultured with the same treatments in vitro from Day 2 to 8. Hyaluronidase-2, but not Hyalovet, enhanced blastocyst formation and reduced inner cell mass apoptosis. Hyalovet inhibited hatching. In conclusion, the presence of large-size HA (500–750 kDa) in the vicinity of developing embryos appears to disturb the oviductal environment and embryo development in vivo and in vitro. In contrast, we show evidence that breakdown of HA into smaller fragments is required to maximize embryo development and blastocyst quality

Expression of ghrelin and Its receptor mRNA in ovibne oocyte and cumulus cells

Energy balance is regulated by ghrelin which is a neuroendocrine modulator. Ghrelin is expressed in reproductive organs. However, the role of ghrelin during in vitro maturation (IVM) and bovine preimplantational development is limited. The purpose of this study was to measure the expression of ghrelin (GHRL) and its receptor growth hormone secretagogue receptor 1A (GHS-R1A) mRNA, and determine cumulus oocyte complex (COC) viability after IVM with 0, 20, 40 and 60 pM of ghrelin. Also, pronuclear formation was recorded after in vitro fertilization (IVF). GHRL and GHS-R1A mRNA expression in oocyte and cumulus cells (CCs) was assessed using reverse transcription-polymerase chain reaction (PCR). Oocyte and CC viability were analyzed with the fluorescein diacetate fluorochrome-trypan blue technique. Pronuclear formation was determined 18 hours after IVF with Hoechst 33342. The results demonstrated that ghrelin mRNA is present in oocyte and CCs before and after 24 hours IVM with all treatments. Ghrelin receptor, GHS-R1A, was only detected in oocytes and CCs after 24 hours IVM with 20, 40 and 60 pM of ghrelin. Oocyte viability was not significantly different (P=0.77) among treatments. However, CC viability was significantly lower (P=0.04) when COCs were matured with ghrelin (77.65, 72.10, 66.32 and 46.86% for 0, 20, 40, and 60 pM of ghrelin, respectively). The chance of two pronuclei forming were higher (P=0.03) when ghrelin was not be added to the IVM medium. We found that ghrelin negatively impacts CC viability and pronuclear formation.Fil: Sirini, Matías Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Quintana, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Artrópodos; ArgentinaFil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Relling, Alejandro Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Anchordoquy, Juan Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentin

Expression of vascular endothelial growth factor in normal and tumoral liver mouse cells

In previous studies have been reported a controversy between the reactivity of mono and polyclonal antibodies in different cells populations of the mouse. In this study we focus on the immunoreactivity of the monoclonal antibody VEGF (C-1) in order to analyze its expression in two cell populations of regenerating mouse liver (hepatocytes and endothelial cells) after partial hepatectomy, and two transplanted hepatocarcinomas (ES2 and SS1K). C3H/S male mice were divided into two groups and kept under standard conditions for circadian periodicity analysis, one group were transplanted with the tumors into the subcutaneous tissue of the animal flank and the other group were subjected to a partial hepatectomy (70 %). The animals were killed by decapitation and exsanguination. Samples of regeneration liver, ES2 and SS1K tumors were processed for histology and immunostained with VEGF (C-1) mouse monoclonal IgG2a antibody. The results showed strongly immunopositivity reaction for VEGF either in hepatocytes or endothelial cells of mouse regenerating liver. We also demonstrated positive immunoreaction in both malignant tumors ES2 and SS1K endothelial and parenchymal cells. We conclude that the use of VEGF (C-1) mouse monoclonal IgG2a antibody can be applied successfully in experimental design leading to study processes involved in mice’s angiogenesisFacultad de Ciencias Médica

Prepartum fatty acid supplementation in sheep. III. Effect of eicosapentaenoic acid and docosahexaenoic acid during finishing on performance, hypothalamus gene expression, and muscle fatty acids composition in lambs

The objectives of this study were to evaluate the effect of feeding an enriched diet with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to finishing lambs born from ewes supplemented either with or without EPA and DHA during late gestation on productive performance, muscle fatty acid (FA), and hypothalamus mRNA concentration of metabolic genes and hormone receptors. Lambs born from dams fed during the last 50 d of gestation either with a control diet containing 0.39% Ca salts of palmitic fatty acid distillate (C) or Ca salts enriched with EPA and DHA (PFA) were used. After weaning lambs (n = 70) were blocked by weight (BW) and used in a 2 × 2 factorial into 2 finishing diets containing 1.5% of C or PFA. The 2 factors were the ewe diet and the finishing diet. Lambs (37.9 ± 0.4 kg) were weighed and blood sampled for glucose and NEFA measurements at days 1, 14, 28, and 42. Dry matter intake (DMI) was measured daily. At day 43, 14 females and 14 males were slaughtered, and hot carcass weight, body wall thickness, rib eye area, and FA composition of Longissumus thoracis muscle were evaluated. Female hypothalamuses were obtained and mRNA concentration of hormone receptors, neuropeptides, and their receptors was measured. Lambs born from PFA dams were heavier (P 0.1). Lambs born from PFA dams had a greater concentration of C22:0 (P < 0.03). Lambs fed C had higher concentrations of C18:1c15 (P < 0.01), C17:0 (P < 0.09), C18:0 (P < 0.09), and n6/n3 (P < 0.01). Lambs fed PFA had greater concentration (P < 0.05) of C16:1, C22:1, C20:5, C22:5, C22:6, total n3 FA, and total EPA and DHA. There was a significant dam × finishing diet interaction (P ≤ 0.08) on mRNA concentration for MCR3, CCK-R, Cort-R, and CART. Lambs, which had the same treatment as their dams, showed lower overall mRNA concentration than those with different treatments between them and their dams. Lambs born from PFA ewes had lower concentration of MCR4 mRNA (P = 0.09) than C. Agouti-related peptides mRNA concentration was lower in lambs fed PFA (P = 0.06) than C. In conclusion, changes on lamb performance, muscle fatty acid composition, and metabolic neuropeptides depend not only on the lamb diet, but also on the dam diet during late gestation.Fil: Carranza Martin, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Coleman, Danielle Nicole. Ohio State University; Estados UnidosFil: Garcia, Lyda Guadalupe. Ohio State University; Estados UnidosFil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Relling, Alejandro Enrique. Ohio State University; Estados Unido

Effect of eicosapentaenoic acid on bovine cumulus–oocyte complex in vitro

The aim of the present study was to investigate the effects of eicosapentaenoic acid (EPA) supplementation during in vitro maturation (IVM) of bovine oocytes. The concentrations tested in all experiments were 1 nM, 1 μM, and 1 mM EPA. The effect of EPA was evaluated on cumulus‐oocyte complexes (COC) by oocyte maturation (cumulus expansion area and oocyte nuclear maturation), genotoxicity [single cell gel electrophoresis (SCGE)], and cytotoxicity (apoptosis, viability, and MTT assays) end points. The maturation parameters were affected by exposure of COC to different EPA concentrations in the IVM medium. Cumulus expansion area increased in the presence of 1 nM EPA (P < 0.05) whereas addition of 1 nM EPA (P < 0.05) decreased cumulus expansion after 24 h of IVM. Moreover, the maturation rate significantly decreased when 1 mM of EPA was assayed (P < 0.001). EPA at 1 nM induced genotoxic and cytotoxic effects on bovine cumulus cells (CC) and primary DNA lesions (P < 0.001). A significant increase in the frequency of apoptotic (P < 0.01) and necrotic (P < 0.001) cells was observed after 24 h of treatment with 1 nM, 1 μM, and 1 mM EPA. Mitochondrial activity was altered with 1 mM EPA (P < 0.001). We inferred that optimal oocyte quality was partially dependent on the presence of adequate EPA concentrations; EPA could be beneficial to improve oocyte quality in the maturation process, because low concentration tested (1 nM EPA) improved cumulus expansion.Instituto de Genética Veterinari

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dosedependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P50.014 for Zn1; P,0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations 'protect' the integrity of DNA molecule and diminish the percentage of apoptotic CC.Fil: Anchordoquy, Juan Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Picco, Sebastian Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Seoane, Analia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Ponzinibbio, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Mattioli, Guillermo Alberto. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Fisiología. Laboratorio de Nutrición Animal; ArgentinaFil: Peral Garcia, Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Cátedra de Citologí, Histología y Embriología ‘‘A’’ ; Argentin