In vitro production of bovine embryos derived from individual donors in the Corral® dish

Background: Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral (R) dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral (R) dish, each donor group in a separate quadrant. Results: In two experiments, the Corral (R) dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral (R) dish used during IVM and IVC than when only used during IVM (12.9% +/- 2.10 versus 22.8% +/- 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination. Conclusions: In the present study, the Corral (R) dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral (R) dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral (R) dish is used during IVM and IVC

The effect of animal handling procedures on the blood non-esterified fatty acid and glucose concentrations of lactating dairy cows

A

Anesthesia in kittens a review of the literature with stress on the possibilities in Belgium

In Belgium, elective surgical procedures in very young small animals are rarely performed. Consequently, most veterinarians have little experience in anesthetizing pediatric patients. This article describes potential problems of anesthesia in kittens, which are mainly linked to their specific anatomy and physiology. Additionally, some practical anesthetic protocols are highlighted. Combinations with ketamine are practical, economical and efficient options.In België worden zelden electieve chirurgische ingrepen uitgevoerd bij zeer jonge kleine huisdieren. Veel dierenartsen hebben dan ook weinig ervaring met de anesthesie van pediatrische patiënten. In dit overzichtsartikel wordt kort ingegaan op de risicofactoren bij de anesthesie van kittens, gezien hun specifieke anatomie en fysiologie. Vervolgens wordt een overzicht gegeven van geschikte anesthetische protocollen. Injectieanesthesie op basis van combinaties met ketamine is een praktische, economische en efficiënte optie.status: publishe

Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure

The feasibility of repeated collection and enzymatic isolation of large numbers of viable primordial and primary follicles from living donor cows were tested. Ovarian cortical biopsies were collected transvaginally by the Biopsy Pick-Up (BPU) device, a modification of an Ovum Pick-Up instrument. Follicles were enzymatically isolated from the retrieved cortical tissue samples, and follicle viability was determined by a live/dead fluorescent assay. Six cows were subjected to BPU once per week during 4 consecutive weeks, and in each BPU session 4 cortical tissue samples were collected per ovary. Over the 4-week trial period, a total of 1443 primordial and primary follicles were collected, 1358 (94%) of which were primordial and 85 (6%) were primary follicles. In each BPU session, an average 60.1 +/- 10.7 (mean +/- SEM) primordial and primary follicles were isolated per cow. The number of follicles varied considerably throughout the trial period and between cows. Statistical analysis of the data, however, did not support the presence of any distinct trends in the follicle yields over time or between cows. A total of 111 enzymatically isolated follicles were analyzed for viability with fluorescent probes. The vast majority of isolated follicles (92.8%) were totally viable. We conclude that the standardized BPU procedure generates sufficiently large numbers of vital primordial and primary follicles, thus validating BPU as a new tool for research into early bovine follicular development.status: publishe

Typical metabolic changes in high producing dairy cows early post partum and their consequences on oocyte and embryo quality

The negative energy balance (NEB) is characterized by typical biochemical changes such as high non-esterified fatty acid (NEFA), high beta-hydroxybutyrate (BHB) and low glucose concentrations. The concentrations of these metabolites were extensively analyzed in the follicular fluid of high yielding dairy cows during NEB and were imitated in in vitro maturation models to investigate their effects on oocyte quality. The studies reviewed in this paper showed that the typical metabolic changes that occur during NEB are well reflected in the follicular fluid (FF) of the dominant follicle. However, the oocyte seems to be relatively isolated from excessively high NEFA or excessively low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that such metabolic changes in the FF associated with a NEB (high NEFA and low glucose concentrations) are indeed toxic for the oocyte, resulting in hampered oocyte maturation, increased apoptosis and necrosis in the cumulus cells, and jeopardized developmental competence of the resulting embryos. Only in moderately hypoglycemic maturation conditions did BHB have an additive toxic effect. These in vitro maturation models, based on in vivo observations and reviewed in this paper, suggest that a period of NEB may hamper the fertility of high yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF, directly affecting oocyte quality. Finally, it was also demonstrated in our lab that the embryo quality of lactating high producing dairy cows is inferior compared to that of non-lactating dairy heifers or beef cows

Effect of organic selenium in the diet on sperm quality of boars

The effect of a diet supplemented with organic selenium (Se) on sperm production and quality of boars was investigated. Sixty mature boars from a commercial artificial insemination centre were randomly allocated at Day (D) 0 into Group A and B. Group A received the regular ration supplemented with inorganic Se (0.4 mg/kg feed as Na2SeO3) whereas Group B was switched to the same diet but with organic Se (0.4 mg/kg fed as Se-yeast). The sperm was investigated during 4 months (D0, D30, D60, D75, D90, D105 and D120). Sperm concentration and motility were objectively measured using a photometer and Computer Assisted Semen Analysis (CASA) respectively. Morphology of the sperm was assessed using eosin-nigrosin staining and the resistance to induction of oxidative stress (production of malonaldehyde, MDA) through thiobarbituric acid reagent species analysis. Additionally, the Se concentration in sperm and blood plasma were measured. Repeated measures analysis of variance (anova) from D60 to 120 (spermatogenesis of approximately 2 months) or anova at D120 (Se concentrations) were used for statistical analysis. The total number of ejaculated sperm was not significantly different between both groups, but boars of Group B had a significantly higher sperm concentration (434.6 vs 514.1 x 106 sperm/ml; p 0.05) were observed for the other parameters. Under the present study conditions, changing from inorganic Se to organic Se in the diet of boars increased sperm concentration but reduced some motility parameters and resistance to oxidative stress

[Four years of ovum pick-up (OPU) and in vitro fertilization (IVF) in Belgian blue donor cows]

Between 1996 and 2000 79 Belgian blue donor cows were submitted to OPU-IVF. They all had a history in classical embryo transfer programs with disappointing results. Two different in vitro embryo production protocols were used. Between 1996 and 1998 (period A), oocytes were matured in M 199 and Epidermal Growth Factor (EGF) and Foetal Calf Serum (FCS) were added. Subsequently, the, oocytes were cultured in a granulosa co-culture system in a Synthetic Oviduct Fluid (SOF) medium. From 1998 until 2000 (Period B), M 199, FCS, equine chorionic gonatrophin (eCG) and granulosa cell co-culture were used for in vitro maturation. Zygotes were subsequently cultured in SOF with a co-culture of bovine oviduct epithelial cells (BOEC). During period A, 531 OPU-IVF sessions were performed, collecting 2111 cumulus oocyte complexes (COCs), of which 928 (44%) were of good morphological quality. The in vitro maturation (IVM), fertilization (IVF) and culture (lVC) resulted in 241 transferable embryos. When fresh embryos (n=88) were transferred, a pregnancy rate of 27% was obtained, whereas the transfer of frozen embryos (n=18) resulted in 29% pregnant recipients. During period B, 1519 OPU sessions were performed, collecting 7027 COCs, with 2157 (31%) of them being of good quality. Following IVM-IVF-IVC, we cultured 1120 transferable embryos of which 438 were transferred fresh, resulting in a pregnancy rate of 39%. The transfer of frozen embryos (n=139) yielded a pregnancy rate of only 5%. The overall results improved over the years, while individual donor variability was one of the main factors that had influence on the OPU-IVF success rate. At the end of the second period, an average of 0.8 embryos were obtained per OPU session. Since freezing of in vitro derived bovine embryos is still problematic, the transfer of fresh embryos remains the best option. However, in our circumstances the availability of good quality recipients appeared to be the limiting factor

The effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality

Obesity is associated with female reproductive abnormalities. Hyperlipidaemia might alter the embryonic micro-environment and potentially result in reduced fertility. We aimed to induce hyperlipidaemia nutritionally and investigate the consequences of hyperlipidaemic culture conditions on bovine in vitro embryo development, embryo quality and gene expression patterns. Bovine zygotes (n = 1545) were cultured in synthetic oviductal fluid medium supplemented with serum from heifers (n = 3), each fed three successive dietary treatments: (i) control serum, following a hay-based diet, (ii) hyperlipidaemic serum, following a carbohydrate and protected palm-oil-rich diet (FatCh) or (iii) hyperlipidaemic serum, following a protected palm-oil-rich diet (Fat). Blastocysts were evaluated for development, cell count, picnotic and mitotic indexes and cryotolerance. Selected mRNA transcripts were measured by quantitative RT-PCR. FatCh and Fat diets approximately doubled the total cholesterol concentrations, compared with controls (167.1 +/- 11.9, 150.0 +/- 12.8 versus 83.4 +/- 13.7 mg/dl, respectively, P < 0.05), and fatty acid concentrations (8146.60 +/- 214.61, 6935.56 +/- 1081.04 versus 3944.0 +/- 425.07 mu mol/l, respectively, P < 0.05). Supplementation of culture media with FatCh and Fat serum significantly reduced blastocyst rates, compared with controls (27.8, 23.4% versus 36.2%, respectively, P < 0.01), total cell number (103.3 +/- 30.1, 95.6 +/- 28.2 versus 146.9 +/- 34.2, respectively, P < 0.01), mitotic index (1.3 +/- 1.1, 1.7 +/- 2.4 versus 3.6 +/- 2.2%, respectively, P < 0.01) and hatching rates after vitrification (20.4, 13.8 versus 35.7%, respectively, P = 0.03). Embryos in FatCh and Fat groups exhibited significantly higher mRNA levels for genes related to apoptosis and metabolism, compared with controls. This combined in vivo and in vitro model indicates that the exposure of preimplantation embryos to hyperlipidaemic conditions may result in reduced embryo quality and developmental potential, possibly resulting in poorer fertility