Zona Pelúcida: una matriz extracelular con aplicaciones en anticoncepción de carnívoros domésticos

Artículo de Publicación ISIZona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. In most mammalian species this matrix consists of three families of glycoproteins likely to suffer several postraductional modifications to acquire different immunological and biochemical properties. The ZP are involved in the initial recognition and binding events of sperm to oocyte investments. Because of its major role in the fertilization process (interaction with sperm, induction of acrosome reaction, control of polyspermy) the ZP has been used as an antigen to induce immunological response in order to produce female infertility, a relevant issue in domestic carnivores. Depending on the predominant immunological response (humoral or cellular) the infertility can be reversible (Immunocontraception) or irreversible (immunosterilization), respectively. Currently the goal is to develop recombinant ZP antigen with immunodominant epitopes; in order to produce predictable and safely response

Temporal expression of GDF-9 and BMP-15 mRNAs in canine ovarian follicles

© 2016 Elsevier Inc. This study aimed to investigate the expression profiles of growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) mRNA in canine oocytes and follicular cells throughout development at the different phases of the estrus cycle. Ovarian structures (follicles and CL) and plasma progesterone concentration were used to confirm the physiological status of each donor. Denuded oocytes and their follicular cells were recovered from follicles (n = 675) distributed into 4 types (preantral, small antral ∼0.2–0.39 mm, medium antral ∼0.4–5.9 mm, and large antral ∼6–8 mm). Total RNA was extracted and reverse transcribed, and the levels of expression for these 2 genes were determined using a quantitative real-time polymerase chain reaction technique; the data were evaluated by ANOVA. Relative expressions levels of GDF-9 and BMP-15 transcripts were detected in the oocyte and follicular cells in all follicular stages evaluated, showing differential change

Zona pellucida: An extracellular matrix with applications in the study of immunocontraception in domestic carnivores

La zona pelúcida (ZP) es una matriz extracelular que rodea al ovocito en los mamíferos y está constituida por tres familias de glicoproteínas en la mayoría de ellos, que difieren en sus propiedades inmunológicas y bioquímicas. La ZP participa en los eventos de reconocimiento y unión del espermatozoide a las envolturas del ovocito, siendo una de sus principales funciones la participación en el proceso de fecundación (interacción con el espermatozoide, inducción de la reacción acrosómica, control de la poliespermia). La ZP ha sido utilizada como antígeno para inducir una respuesta inmune tendiente a disminuir la fertilidad de las hembras, condición que adquiere especial relevancia en el caso de los carnívoros. Se ha observado que dependiendo del tipo de respuesta inmune predominante -humoral o celulares efecto en la fertilidad puede ser reversible (inmunoanticoncepción) o irreversible (inmunoesterilización) respectivamente. En la actualidad el propósito es el desarrollo de antígenos de ZP recombinantes, con epítopes inmunodominantes, con el objeto de inducir una respuesta inmune predecible y segura.444 - 450BimestralZona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. In most mammalian species this matriz consists of three families of glycoproteins likely to suffer several postraductional modifications to acquire different immunological and biochemical properties. The ZP are involved in the initial recognition and binding events of sperm to oocyte investments. Because of its major role in the fertilization process (interaction with sperm, induction of acrosome reaction, control of polyspermy) the ZP has been used as an antigen to induce immunological response in order to produce female infertility, a relevant issue in domestic carnivores. Depending on the predominant immunological response (humoral or cellular) the infertility can be reversible (Immunocontraception) or irreversible (immunosterilization), respectively. Currently the goal is to develop recombinant ZP antigen with immuneodominant epitopes in order to produce predictable and safely response

Description of some seminal parameters in german sheperd dog

Con la finalidad de estimar algunos parámetros seminales y sus variaciones de acuerdo a la edad del reproductor, se estudiaron 100 eyaculados de 25 ejemplares de raza Ovejero Alemán, cuyas edades fluctuaban de 1 a 5 años. Las muestras de semen se extrajeron mediante vagina artificial, una vez al mes durante el período otoño-invierno. Luego de la recolección cada eyaculado fue eva­luado inmediatamente a través de métodos rutina­rios. Se obtuvieron los siguientes promedios gene­rales (± D.S.): volumen 22,91 ± 14,47 ml; pH 7,04 ± 0,28; motilidad 65 ±29,9%, concentra­ción espermática 87,45 ± 65,15 x 106 espermato­zoides por ml; espermatozoides totales por eyacu­lado 1,53 ± 1,02 x 109 y anormalidades totales 7,70 ± 3,70%. La mayoría de las muestras presen­taron color blanco brillante (95%) de tipo acuoso (99%). Solamente la cantidad total de espermato­zoides por eyaculado mostró diferencias significati­vas entre las distintas edades (P < 0,05)

Pannexin channels increase propidium iodide permeability in frozen-thawed dog spermatozoa

Pannexins (Panx) are proteins that form functional single membrane channels, but they have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen–thawed dog spermatozoa using flow cytometry and immunofluorescence analyses, evaluating the relationship of these proteins with propidium iodide (PI) in frozen–thawed spermatozoa. Fresh and frozen–thawed dog spermatozoa from eight dogs were preincubated with 3 mM PI with or without 15 mM carbenoxolone (CBX) or 1 mM probenecid (PBD), two Panx channel inhibitors, and then incubated with rabbit anti-Panx1, anti-Panx2 and anti-Panx3 antibodies (1 : 200). Panx immunolocalisation was assessed by fluorescence microscopy. Flow cytometry data were evaluated by analysis of variance. All three Panx proteins were found in dog spermatozoa: Panx1 was mostly localised to the acrosomal and equatorial segment, Panx2 was found in the posterior region of the head and tail and Panx3 was localised to the equatorial and posterior head segment. The percentage of PI-positive cells determined by flow cytometry was reduced (P , 0.05) in the presence of Panx inhibitors. These results show that Panx proteins are present in dog spermatozoa and increase PI permeability in frozen–thawed dog sperm, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells

GDF-9 and BMP-15 mRNA levels in canine cumulus cells related to cumulus expansion and the maturation process

Simple Summary The knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte-cumulus cells' regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation. Abstract The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) FONDECYT 1171670 VID, University of Chile YV029/01-1

Description of reproductive characteristics of three Brucella canis seropositive dogs. Descripción de características reproductivas en tres perros seropositivos a Brucella canis

Seminal and histological reproductive characteristics in three Brucella canis seropositive dogs are described. Seminal volume and sperm morphology were altered and no sperm was seen in one dog. This agrees with histological findings where spermatozoid development was altered and eritrocites inside tubular lumen were seen indicating hemo-testicular barrier failure. These results confirm the negative impact of the disease on reproductive performance with subsequent infertility

In vitro sperm penetration through the zona pellucida of immature and in vitro matured oocytes using fresh, chilled and frozen canine semen

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1–10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p < 0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p < 0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P < 0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. Therewas a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10 h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.This research was supported by Grants ENL 05/8 DID and FONDECYT 1060602 to M.D.R

Selection of frozen bull spermatozoa for in vitro fertilization Selección de espermatozoides de toro para fecundación in vitro

The swim-up and Percoll gradient techniques were evaluated for the selection of frozen bovine spermatozoa to be used on an in vitro fertilization system. The parameters assayed were: concentration, morphology, motility and rate of in vitro fertilization and rate of development. The spermatozoa were from a single bull and the oocytes were aspirated from ovaries obtained at the slaughter house and matured in vitro. Sperm motility increased significantly (p&lt;0.05) from 70%-75% to 90% - 95%. However after sperm selection there was no significant difference between both methods (p&gt;0.05). Sperm concentration after swim-up was 3.33 × 106 sp/ml and with Percoll 16.5 × 106 sp/ml (p&lt;0.01). Samples obtained by both methods showed a decrease in abnormal spermatozoa as compared to controls (17.3% in swim-up; 24.8% in Percoll and 47% in control) (p&lt;0.01). There was a significant difference between both methods only in regards to anomalies of the neck region (3.5% in swim-up and 9.6% in Percoll (p&gt;0.

Evaluation of glucose as a cryoprotectant for boar semen

Artículo de Publicación ISIFertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111.0mM Tris, 31.4mM citric acid, 185.0mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200mM Tris; 70.8mM citric acid, 55.5mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200mM Tris, 70.8mM citric acid, 55.5mM fructose, 20 per cent egg yolk 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 10(6) sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30degreesC/minute to - 196degreesC. The straws were thawed at 60degreesC for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively, these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively