Copper sulfate and sodium selenite lipid-microencapsulation modifies ruminal microbial fermentation in a dual-flow continuous-culture system.

Undesirable interactions between trace mineral elements and ruminal contents may occur during digestion when mineral salts are supplemented. Antimicrobial effects of copper sulfate (CuSO4) may affect ruminal digestibility of nutrients when fed as a source of copper (Cu), while sodium selenite (Na2SeO3) may be reduced in the rumen to less available forms of selenium (Se). Our objective was to evaluate if protection of CuSO4 and Na2SeO3 by lipid-microencapsulation would induce changes on ruminal microbial fermentation. We used 8 fermentors in a dual-flow continuous-culture system in a 4 × 4 duplicated Latin square with a 2 × 2 factorial arrangement of treatments. Factors were CuSO4 protection (unprotected and protected by lipid-microencapsulation) and Na2SeO3 protection (unprotected and protected by lipid-microencapsulation). Treatments consisted of supplementation with 15 mg/kg of Cu and 0.3 mg/kg of Se from either unprotected or protected (lipid-microencapsulated) sources, as follows: (1) Control (unprotected CuSO4 + unprotected Na2SeO3); (2) Cu-P (protected CuSO4 + unprotected Na2SeO3); (3) Se-P (unprotected CuSO4 + protected Na2SeO3); (4) (Cu+Se)-P (protected CuSO4 + protected Na2SeO3). All diets had the same nutrient composition and fermentors were fed 106 g of dry matter/d. Each experimental period was 10 d (7 d of adaptation and 3 d for sample collections). Daily pooled samples of effluents were analyzed for pH, NH3-N, nutrient digestibility, and flows (g/d) of total N, NH3-N, nonammonia N (NAN), bacterial N, dietary N, and bacterial efficiency. Kinetics of volatile fatty acids was analyzed in samples collected daily at 0, 1, 2, 4, 6, and 8 h after feeding. Main effects of Cu protection, Se protection, and their interaction were tested for all response variables. Kinetics data were analyzed as repeated measures. Protection of Cu decreased acetate molar proportion, increased butyrate proportion, and tended to decrease acetate:propionate ratio in samples of kinetics, but did not modify nutrient digestibility. Protection of Se tended to decrease NH3-N concentration, NH3-N flow, and CP digestibility; and to increase flows of nonammonia N and dietary N. Our results indicate that protection of CuSO4 may increase butyrate concentration at expenses of acetate, while protection of Na2SeO3 tended to reduce ruminal degradation of N. Further research is needed to determine the effects of lipid-microencapsulation on intestinal absorption, tissue distribution of Cu and Se, and animal performance.

Comparison of two inoculation methods for Microsporum canis culture using the toothbrush sampling technique.

BACKGROUND: The fungal culture toothbrush method is a common method for obtaining material for fungal cultures to diagnose dermatophytosis. The optimal technique for inoculation onto the agar surface has not been studied. HYPOTHESIS/OBJECTIVES: To compare two inoculation techniques; the first involved pressing the toothbrush onto the plate surface (Procedure A) and the second involved pressing the toothbrush onto the agar, as well as transferring hairs and scales entrapped in the bristles. (Procedure B). The hypothesis was that transferring hairs onto the plate would increase the likelihood of obtaining positive cultures. ANIMALS: Twenty-six cattery-housed cats were sampled using the toothbrush technique. Two toothbrush samples were obtained from each cat. METHODS AND MATERIALS: The two toothbrush samples from each cat were randomized to Procedure A or B, and the investigator was blinded to inoculation technique. Cultures were performed on a medium specific for dermatophytes. The number of positive plates, and the presence and abundance of colonies of dermatophytes and contaminant moulds were compared between the two techniques. RESULTS: Twenty-one cats were culture-positive for Microsporum canis. Procedure A resulted in a significantly higher number (P < 0.01) of positive plates (20 of 21; 95%) compared with Procedure B (seven of 21; 33%). These results were due mainly to higher plate invasion by contaminant moulds, using Procedure B. CONCLUSIONS AND CLINICAL IMPORTANCE: Based upon the findings of this study, the optimum inoculation technique is to press toothbrush bristles onto agar plates to maximize growth of M. canis and minimize introduction of contaminant inoculation.

Assessment of two different types of sample for the early detection and isolation of thermophilic Campylobacter in broiler farms.

In order to assess the optimal method for the early detection and isolation of thermophilic Campylobacter in broilers at farm level, two types of samples were compared: caecal contents obtained by necropsy and cloacal swabs transported in charcoal Amies medium. The study was conducted in five batches of broilers from five different farms, where weekly samples (caecal contents and cloacal swabs) from 30 birds were obtained. Samples were plated onto selective agar (modified charcoal cefoperazone desoxycholate agar, mCCDA) for Campylobacter isolation. Four out of five batches were positive for Campylobacter. No marked differences in sensitivity of both sample types were observed. However, a higher percentage of positive birds were detected when cloacal swabs were used. The results show that cloacal swab samples are adequate, and in some cases even better than caecal samples for the early detection of Campylobacter in broiler flocks at farm level. Also, this sample avoids sacrificing birds to test Campylobacter, which not only allows saving time in sample collection, transportation and processing at the laboratory, but also improves bird welfare and cost of sampling.

Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV).

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10(5) plaque-forming units (PFU) mL(-1) VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory-reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10(3) PFU mL(-1) ) than among groups that survived exposure to VHSV (1.0-2.9 × 10(2) PFU mL(-1) ); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39-47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.

Establishment of culture conditions for survival of Histomonas meleagridis in transit.

Fresh ceca samples from turkeys in North Carolina infected with Histomonas meleagridis were collected at necropsy, inoculated into warmed Dwyers medium, and sent by overnight courier to our laboratory at The University of Georgia. Further incubation at 40 C yielded positive cultures from all four samples. PCR and DNA sequencing confirmed the presence of H. meleagridis. To further establish conditions for survival in transit, we infected turkeys with H. meleagridis, euthanatized the birds 10 days postinfection, and allowed carcasses to incubate at room temperature for either 2 or 24 hr. After incubation, samples of cecal contents (0.5 g) were placed in Dwyers medium and held at 4, 25, or 30 C for 6, 18, 24, 48, 72, 96, or 120 hr, simulating holding conditions during transit. Samples were placed in a 40 C incubator at the specified times and examined daily for histomonad growth by light microscopy. Positive histomonad growth was detected from cecal samples obtained from the 2-hr incubated carcass and from cultures held at 30 C for 6, 18, 24, 48, and 72 hr. No growth was seen from cultures held at 25 or 4 C or at any temperature from the carcass allowed to incubate for 24 hr at room temperature. These results suggest that positive isolation can be made from field samples, provided that material is collected at warm temperatures and transported rapidly to the laboratory.

Comparison of carpet and toothbrush techniques for the detection of Microsporum canis in cats.

OBJECTIVES: The aim of this study was to evaluate the diagnostic concordance between the toothbrush and carpet techniques for the detection of Microsporum canis in cats in a field study. METHODS: Thirty-nine Persian cats from a cattery were used. Fungal culture samples from the haircoat of each cat were collected by stroking the coat with a sterile toothbrush and a 5 × 5 cm-sized sterile carpet square (n = 78 total samples). Specimens were inoculated onto Mycosel Agar and incubated at 25°C for 21 days. Both techniques were compared using the following parameters: number of plates without fungal growth, number of plates with contaminant growth and number of plates positive for dermatophytes. RESULTS: The feline population in the study cattery was 39. Thirty (77%) were symptomatic and nine (23%) asymptomatic. The diagnosis was made via carpet and toothbrush methods and 78 cultures were performed. On day 21, M canis was detected in all culture plates. No contaminant molds were observed. CONCLUSIONS AND RELEVANCE: The concordance rate between the carpet and toothbrush techniques among the 78 evaluable culture plates was 100%. Both methods are equally effective for collecting material for Mcanis culture. Additionally, both techniques are inexpensive and easy to perform in feline clinical practice.

Use of cefovecin in dogs and cats attending first-opinion veterinary practices in Australia.

BACKGROUND: Cefovecin is a long-acting third-generation cephalosporin commonly used in veterinary medicine. Third-generation cephalosporins are critically important antimicrobials that should only be used after culture and susceptibility testing. The authors describe the common indications for cefovecin use in dogs and cats, and the frequency of culture and susceptibility testing. MATERIALS AND METHODS: A cross-sectional study was performed using clinical records extracted from VetCompass Australia. A previously described method was used to identify records containing cefovecin. The reason for cefovecin use was annotated in situ in each consultation text. RESULTS: Over a six-month period (February and September 2018), 5180 (0.4 per cent) consultations involved cefovecin administration, of which 151 were excluded. Cats were administered cefovecin more frequently than dogs (1.9 per cent of cat consultations and 0.1 per cent of dog consultations). The most common reasons for cefovecin administration to cats were cat fight injuries and abscesses (28 per cent) and dermatitis (13 per cent). For dogs, the most common reasons for cefovecin administration were surgical prophylaxis (24 per cent) and dermatitis (19 per cent). Culture and susceptibility testing were reported in 16 cases (0.3 per cent). CONCLUSION: Cefovecin is used in many scenarios in dogs and cats where antimicrobials may be either not indicated or where an antimicrobial of lower importance to human health is recommended.

Development and assessment of a novel ex vivo corneal culture technique involving an agarose-based dome scaffold for use as a model of in vivo corneal wound healing in dogs and rabbits.

OBJECTIVE: To develop and assess a novel ex vivo corneal culture technique involving an agarose-based dome scaffold (ABDS) for use as a model of in vivo corneal wound healing in dogs and rabbits. SAMPLE: Corneas from clinically normal dogs (paired corneas from 8 dogs and 8 single corneas) and rabbits (21 single corneas). PROCEDURES: 8 single dog corneas (DCs), 1 DC from each pair, and 10 rabbit corneas (RCs) were wounded with an excimer laser; 1 DC from each pair and 11 RCs remained unwounded. Corneas were cultured for 21 days on ABDSs (8 pairs of DCs and all RCs) or on flat-topped scaffolds (8 single DCs). The surface area of corneal fluorescein retention was measured every 6 (DCs) or 12 (RCs) hours until full corneal epithelialization was detected. Changes in corneal clarity were evaluated at 0, 7, 14, and 21 days. RESULTS: Median time to full epithelialization for wounded dog and rabbit corneas was 48 and 60 hours, respectively; among wounded DCs, time to full epithelization did not differ by scaffold type. After 21 days of culture on ABDSs, all DCs and RCs that epithelialized developed a circular, diffuse, cloud-like pattern of optical haze, whereas DCs cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze. All corneas on the ABDSs maintained convex curvature throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Wounded ex vivo DCs and RCs cultured on ABDSs reliably epithelialized, formed optical haze (consistent with in vivo wound healing), and maintained convex curvature. This culture technique may be adaptable to other species.

Hemolytic activity of Trichomonas gallinae isolates does not correspond with clinical virulence.

The hemolytic activity of 22 Trichomonas gallinae isolates was investigated using an 18h erythrocyte hemolysis assay which has been shown to correlate with the clinical virulence of T. vaginalis. Absorbance of the assay supernatants was measured at 540nm and expressed as percentage of complete hemolysis. Mean hemolytic activity of the T. gallinae isolates ranged from 3.5% to 53.4% and did not correspond with clinical virulence. The results of this investigation suggest hemolytic activity is not a useful in vitro virulence assay for T. gallinae.

Beta-carotene is incorporated or mobilized along with triglycerides in bovine adipose tissue in response to insulin or epinephrine.

Pasture fed cattle ingest substantial amounts of beta-carotene (beta-C). Not all of the carotenoid compound is transformed into vitamin A, but the surplus is deposited in adipose tissue (AT). The mechanisms of beta-C incorporation and mobilization are unknown. Two experiments were conducted using explants from bovine AT cultured in vitro. First, beta-C incorporation by explants from three animals was examined with different beta-C concentrations (0, 1, 5 and 20 microm) and different times of incubation (every 5 h up to 25 h). The data showed a significant increase of beta-C concentration in explants only for 20 microm beta-C. Secondly, effects of insulin and epinephrine on beta-C and triglyceride (TG) contents of explants were studied. Explants from six animals were incubated with either hormone and 0 or 20 microm beta-C for 20 h. Both TG and beta-C contents were affected positively by insulin and negatively by epinephrine. Interestingly, changes in ratios of beta-C/TG (hormone vs. control) were similar (1.7 x 10(-3) and 1.8 x 10(-3)), respectively, for insulin and epinephrine, indicating that beta-C level is directly related to TG content. We also report the presence of mRNA for beta-C 15, 15' oxygenase in bovine AT. The in vitro culture system using explants from bovine AT is a promising model to investigate factors that might affect the accumulation and metabolism of beta-C.

Use of fetuin before and during vitrification of bovine oocytes.

After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10-30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production.

Diagnosis of Avian Mycoplasmas: A Comparison between PCR and Culture Technique.

Mycoplasma gallisepticum and Mycoplasma synoviae are the causative agents of avian mycoplasmosis in commercial poultry. Among the available tools, polymerase chain reaction (PCR) and culture are confirmatory tools for the diagnosis of mycoplasmosis after the initial serological screening of suspected birds. Overall, 181 samples were analyzed, 152 (84%) and 103 (57%) of which were found positive by PCR and culture, respectively. Further, 54 (92%) broiler samples were found positive for general avian mycoplasma. Among the total positive samples, MS positivity was as high as 72 (47%) by PCR, while it was 45 (44%) by culture. MG positivity was 23% and 25% in PCR- and culture-positive samples. MG grows more easily compared to MS. The agreement value between the tests was 67%. Overall, flock wise prevalence was not much varied. The prevalence of mycoplasmosis was higher during winter. Our study confirmed that PCR is the most sensitive and reliable tool for the diagnosis of avian mycoplasmosis in field samples.

Towards European harmonisation of contagious equine metritis diagnosis through interlaboratory trials.

The performance of culture and PCR methods routinely used to diagnose contagious equine metritis (CEM) was evaluated and compared by two interlaboratory trials involving a total of 24 European laboratories, including 22 National Reference Laboratories for CEM. Samples were swab specimens artificially contaminated with bacteria present in the genital tract of Equidae, some with and some without Taylorella equigenitalis, the causative agent of CEM, and T asinigenitalis, responsible for possible misidentification as T equigenitalis Throughout both interlaboratory trials, PCR performed better in terms of specificity and sensitivity than the culture method, supporting the assertion that PCR should be accepted for CEM diagnosis. However, the culture performance during the second interlaboratory trial was better than during the first one, suggesting that the expertise of participants improved. This reveals the advantage of regular interlaboratory trials to constantly improve the expertise of laboratories. It also shows the need to develop new culture media that are more selective and/or better geared to the metabolism of T equigenitalis in order to improve the bacteriological diagnosis of CEM.

Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.

Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid.

Cultivation of sponge larvae: settlement, survival, and growth of juveniles.

The aim of this study was to culture sponge juveniles from larvae. Starting from larvae we expected to enhance the survival and growth, and to decrease the variation in these parameters during the sponge cultures. First, settlement success, morphological changes during metamorphosis, and survival of Dysidea avara, Ircinia oros, Hippospongia communis, under the same culture conditions, were compared. In a second step, we tested the effects of flow and food on survival and growth of juveniles from Dysidea avara and Crambe crambe. Finally, in a third experiment, we monitored survival and growth of juveniles of D. avara and C. crambe transplanted to the sea to compare laboratory and field results. The results altogether indicated that sponge culture from larvae is a promising method for sponge supply and that laboratory culture under controlled conditions is preferred over sea cultures in order to prevent biomass losses during these early life stages.

In vitro assay to select rainbow trout with variable resistance/susceptibility to viral haemorrhagic septicaemia virus.

The merit of a candidate criterion of resistance to viral haemorrhagic septicaemia virus (VHSV) was tested with the view of producing experimental trout progeny with a predictable level of resistance. The criterion, the measure of in vitro viral replication in excised fin tissue (VREFT) was previously developed. Three experiments were performed, using both ordinary and homozygous doubled-haploid breeders. A set of 48 progeny was tested. Breeders were individually scored for repeated measures of VREFT, and the progeny were tested against VHSV (strain 07-71, serotype 1) through a waterborne challenge (5 x 10(4) pfu ml(-1) during 2 h). Analysis of repeated measures of VREFT revealed the risk of identifying 'false' resistant individuals. The highest value should be considered the most predictive of the resistance status. Survival of progeny ranged from 0 to 100% according to the group and the experiment. The survival was correlated to the mean VREFT value of the breeders in Expts 1 and 2 (R = 0.96 and 0.61 respectively), but not in Expt 3 (R = 0.36, ns) where all tested progeny were highly susceptible. Results thus indicate that viral growth in fin tissue is genetically correlated to resistance to waterborne disease and may be used to produce selected progeny, at least at the experimental scale. Possible implications of the relationship between VREFT and resistance for the study of resistance mechanisms are discussed.

Effects of time at suboptimal pH on rumen fermentation in a dual-flow continuous culture system.

Ruminal pH varies considerably during the day, achieving values below 6.0 when cows consume large amounts of concentrates. Low ruminal pH has negative effects on ruminal fermentation. However, previous studies have indicated that rumen bacteria may resist short periods of low ruminal pH, and it is not clear how long this period may be before rumen microbial fermentation is negatively affected. Seven dual-flow continuous culture fermenters (1,320 mL) were used in 3 replicated periods with the same diet (97 g of dry matter/d of a 60:40 forage-to-concentrate diet, 18.3% crude protein, 35.9% neutral detergent fiber), temperature (39 degrees C), and solid (5%/h) and liquid (10%/h) dilution rates to study the effects of increasing time at suboptimal pH on rumen microbial fermentation and nutrient flow. Treatments were a constant pH of 6.4 and 6 different intervals of time during the day (4, 8, 12, 16, 20, 24 h) at suboptimal pH (5.5), with the rest of the day being at pH 6.4. Polynomial equations were derived using the Mixed procedure of SAS, and linear, quadratic and cubic terms were left in the equation if P < 0.10. True organic matter digestion decreased with increasing time at suboptimal pH and was best described by a cubic regression (TOMD = 58.5 - 2.15x + 0.16x2 -0.0037x3; R2 = 0.74). Digestion of NDF (DNDF = 55.1 - 1.00x; R2 = 0.75) and digestion of ADF (DADF = 56.2 - 1.33x; R2 = 0.78) decreased linearly with increasing time at suboptimal pH. Total VFA had a cubic response (VFA = 112.7 - 2.09x + 0.17x2 - 0.0054x3; R2 = 0.82). The proportion of acetate decreased linearly (acetate = 58.7 - 0.61x; R2 = 0.79). The propionate proportion increased (propionate = 17.6 + 2.09 x -0.044x2; R2 = 0.85) and branched-chain VFA decreased (BCVFA = 4.45 -0.51x + 0.014x2; R2 = 0.75) quadratically. The ammonia N concentration (NH3-N = 5.85 - 0.13x; R2 = 0.46) and flow (NH3-N flow = 0.18 - 0.0039x; R2 = 0.43) decreased linearly as the time at suboptimal pH increased. Crude protein degradation (CPd = 41.9 - 1.60x + 0.060x2; R2 = 0.71), efficiency of microbial protein synthesis (EMPS = 26.6 - 0.33x + 0.021x2; R2 = 0.77), microbial N flow (MN flow = 1.38 - 0.036x + 0.0015x2; R2 = 0.77), and dietary N flow (DN flow = 1.49 + 0.041x - 0.0015x2; R2 = 0.65) had a quadratic response. The flow of essential, nonessential, and most individual AA increased linearly with increasing time at suboptimal pH. The effects of pH on rumen fermentation appear to start as soon as pH drops to suboptimal pH.

The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations.

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.

Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas.

We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5).