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+)

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

Veterinary Science in Catalonia: 1996-2002

Research in veterinary science in Catalonia can be considered a relatively recent activity since the main public entities dedicated to it were developed during the last few decades. The Faculty of Veterinary Science at the Autonomous University of Barcelona (UAB) was created in 1982; the specialization animal husbandry at the School of Agricultural Engineering of the University of Lleida (UdL), in 1983; and the Institute for Food and Agricultural Research and Technology (IRTA) in 1985. Nowadays, the field of veterinary science includes, besides animal medicine and health, topics more related to the economic importance of animals and the hygiene and quality of animal-derived products. This report classifies veterinary science according to its different thematic areas: Animal Health, Animal Anatomy, Animal Medicine and Surgery, Animal Production, Animal Nutrition and Bromatology, and Animal Physiology and Pharmacology. Most of these areas are dealt with in the departments of the Faculty of Veterinary Science of the UAB. Given the economic importance of Animal Production in Catalonia, it has become the domain of the largest public entities (UAB, IRTA, and UdL). The Animal Health Research Center (CReSA) ­an autonomous institution of the UAB and the IRTA­ has operated since 2000. The methodology used in this report was mostly based on the analysis of data obtained from these institutions. Since the report takes into account a small number of both public institutions and people, the data obtained were thoroughly analyzed and classified. All research indicators have been sorted by topic and by the institutions involved. Finally, for each indicator, a global analysis of all areas and organizations is made and compared to the results obtained in the previous report, which comprised the years 1990­1995. The analyzed indicators, recommended by the Institute for Catalan Studies (IEC), are the following: a) The research system. In this section, references to public institutions, centers, departments, and units that carry out veterinary science research in Catalonia are made. b) Lines of research. The different subjects of research that are in the focus of the different areas and institutes are described. The subjects of research and the groups that the General Directorate of Research (DGR) considers as consolidated research groups have been specified. This section also describes research services created by universities and investigation centers to support the research demands of the public and private sectors. c) Human resources. Personnel dedicated to research are classified for every area and institution according to: staff investigator, hired investigator, technical staff, hired technical staff, and intern. The new figures for researchers in the Ramon y Cajal and the Catalan Institute for Research and Advanced Studies (ICREA) programs are specified for every subject. Based on an organizational chart that lists all the personnel comprising the different categories for 2002, we can conclude that: 35% are staff researchers, 17% are hired researchers, 27% technical staff, and 21%, interns. Compared to the period 1990­1995, there was no significant variation in the number of technicians or researchers, only an increase in the number of interns. d) Research funding. This has been classified in competitive projects or projects subject to scientific evaluation, and agreements or non-competitive projects. The funding for competitive projects has been classified according to the source of the program: European, Spanish, or the Government of Catalonia. For non-competitive projects, it is specified whether they were commissioned by the private sector or by public institutions. From the information presented in this section, we can conclude that, for all areas and institutions, the main source of funding is the private sector, indicating a strong projection of veterinary science research in the various productive sectors. For competitive projects, the most important funding are public. If we compare it with the period 1990­1995, we see that the financing for competitive projects in the previous period came to 1.4 million Euros, and that in the current period this sum adds up to 6.47 million Euros. Financing for agreements in the previous period was 1.63 million Euros and during the current period this sum increased to 13.11 million, 9 of which were provided by the private sector. e) Scientific output. This section exclusively considers research results published in scientific journals, books, reports, and theses. The results obtained regarding scientific transference and research services, and other results obtained through agreements have not been included, despite our awareness of their great importance. The development experienced by all areas and institutions indicates a growing interest in the dissemination of results through journals indexed at the ISI (Institute for Scientific Information) register rather than through other formats: 63.7% of the publications published during the period of study were articles that appeared in these journals ­a trend that continues to grow. Compared to the period 1990­1995 period, the number of articles in the Science Citation Index (SCI) increased from 265 to 800. When we compare the scientific output of the world, Spain, Denmark, and the Netherlands, the following conclusions can be drawn: Spain contributes 4.3% of worldwide output, and Catalonia represents 18.7% of the State's total. During the previous period, these figures were 3.1% and 13.5%, respectively. The Netherlands and Denmark, respectively, publish 4.3 and 2.6 times more than Catalonia. To verify the quality of the publications, the number of citations received by published articles was analyzed. The conclusion is that the average number of citations of articles published in Catalonia is equal to the average of the world and Spain, but lower than the number of citations of articles published in the Netherlands and Denmark. In conclusion, veterinary science research in Catalonia has experienced a strong growth in funding, scientific output, and personnel training, despite the fact that the number of researchers and technicians has remained the same. Research teams have consolidated and have focused their efforts on those areas of research that are of greatest interest to the productive sector. This has resulted in more than half of research funding coming from agreements with the private sector. Compared to countries such as Denmark and the Netherlands, scientific output and quality in Catalonia are significantly lower. If public financing for innovative scientific research projects increased, surely these differences would in turn decrease

El altramuz: cultivo e importancia en la alimentación de ovejas

Se realiza una revisión bibliográfica sobre el cultivo del altramuz y especialmente sobre la influencia del consumo de altramuces antes de la cubrición (flushing), sobre los caracteres reproductivos de las ovejas. Se estudian los factores que modifican dicha respuesta como la cantidad de altramuces consumida, la duración de la oferta, la edad de la oveja y la época del ano en que se realiza el «flushing»

Cuantificación de la producción vegetal

A bibliographic review of various methods of estimating fitomass has been carried out. Methods that do not require complicated equipment to predict production are considered as useful, and particularly those methods for the study of shrub plants based on measurements taken from plants in their natural habitat.Se realiza una revisión bibliográfica de los distintos métodos de estimación de la fitomasa, de la que se desprende la utilidad de los métodos que no requieren un equipo complicado para la predicción del rendimiento, siendo especialmente útiles para el estudio de flora arbustiva los basados en medidas obtenidas sobre las plantas en su hábitat natural

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