Producció in vivo versus in vitro d'embrions caprins

Les tecnologies per a la reproducció assistida, com són la inseminació artificial i l'ovulació múltiple i la posterior transferència embrionària (MOET), s'han utilitzat en el cabrum per incrementar l'eficiència reproductiva dels mascles i accelerar el guany genètic de les femelles. No obstant això, el MOET presenta una sèrie de limitacions a causa de: a) variabilitat en la resposta de les femelles al tractament hormonal, b) fecundacions fallides i c) regressió prematura dels cossos lutis. La producció in vitro d'embrions (PIVE) permet superar les limitacions de la tecnologia MOET. La PIVE involucra tres processos: la maduració in vitro dels oòcits, la fecundació in vitro amb espermatozoides capacitats i el cultiu in vitro dels embrions fins a l'estadi de blastocist, moment en què poden ser transferits a cabres receptores o crioconservats per a futurs usos. A més, la recuperació d'oòcits de femelles vives selectes mitjançant laparoscòpia i la reproducció de femelles prepúbers permeten una elevada difusió de les cabres d'alt valor. Així, la PIVE en el cabrum és una font excel·lent i de baix cost d'embrions, que es podran utilitzar tant per a la recerca bàsica en la biologia del desenvolupament com per a aplicacions comercials per produir transgènics i clònics.Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain in male and females, respectively. The current limitations of MOET are due to: i) female variability response to hormonal treatment, ii) fertilization failures and iii) premature regression of corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three mains steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos until blastocyst stage that can be transferred to recipient females or cryopreserved for future use. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU), and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a high diffusion of valuable goats. Also, IVP of goat embryos will provide an excellent source of low cost

Current Status of In Vitro Embryo Production in Sheep and Goats

Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi-step methodology comprising the following procedures: (i)In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co-incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-free genetic movement and easier and cheaper germplasm com- mercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP Research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus-oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes

Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats

In goats, embryo oocyte competence is affected by follicle size, regardless of the age of the females. In previous studies, we found differences in blastocyst development between oocytes coming of small ( 3 mm) in prepubertal (1-2 month-old) goats. Oocyte competence and follicular fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze the fatty acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-nuclear magnetic resonance (1H-NMR) spectrometry. The results showed important differences between adult and prepubertal follicles: a) the presence of α,β-glucose in adult and no detection in prepubertal; b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal; c) the percentage of linolenic acid, total saturated fatty acids and n-3 PUFAs were higher in adults; and d) the percentage of linoleic acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs:n-3 PUFAs ratio were higher in prepubertal goats. No significant differences were found in the follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulusoocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats

Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+)

Produksi Nata Fruticans Dari Nira Nipah

Nipah (Nypa fruticans Wurmb.) menghasilkan nira yang dapat diperoleh melalui penyadapan tandan buah. Dalam keadaan segar nira nipah memiliki rasa manis karena mengandung gula yang cukup tinggi. Cairan ini merupakan media yang subur bagi pertumbuhan mikroorganisme, sehingga nira nipah berpotensi digunakan sebagai bahan baku untuk menghasilkan produk melalui proses fermentasi seperti nata. Nata merupakan jenis pangan yang dikelompokkan sebagai makanan penyegar atau pencuci mulut. Percobaan produksi nata fruticans dilakukan melalui proses fermentasi nira nipah yang masih segar dengan perlakuan penambahan gula 0, 50, 75 dan 100 g per liter nira nipah. Produksi nata fruticans dari nira nipah segar yang ditambahkan gula pada berbagai kadar diperoleh rendemen antara 76,52%-90,97% atau rata-rata 86,05%. Penambahan gula pada nira nipah segar berpengaruh tidak nyata terhadap rendemen produksi nata fruticans. Penggunaan nira nipah segar dengantanpa penambahan gula menghasilkan nata fruticans dengan rendemen rata-rata 83,74%

Ús del mètode puzzle de treball cooperatiu a l'assignatura d'Etnologia per a la identificació racial

Les jornades d'innovació docent a la Facultat de Veterinària tenen per objectiu l'intercanvi d'experiències docents entre el professorat de la Facultat com a punt de partida per reflexionar sobre l'estat actual de la docència i estimular la inclusió de noves activitats d'innovació als estudis de grau. Aquestes jornades estan organitzades pel Grup d'Innovació Docent de la Facultat de Veterinària amb el suport de la Unitat de Formació i Innovació Docent (OQD) de la Universitat Autònoma de Barcelona

Effect of Roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 m M of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 m M of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 m M ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 m M (45.2, 36.1 and 39.4%, respectively), however the percentage of lastocysts was higher in the control group. Western blot for the MAPK and p34 cdc2 showed that both enzymes were active in prepubertal goat oocytes after 24 h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development