The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Isolation of Canine Mammary Cells With Stem Cell Properties and Tumour-Initiating Potential

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.     

Influence of the Cumulus and Gonadotropins on the Metabolic Profile of Porcine Cumulus–Oocyte Complexes During In Vitro Maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A₁ (with a dense cumulus), A₂ (with a translucent cumulus), B₁ (with the corona radiata), B₂ (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A₁ COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.     

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus–Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus–oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm³) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29–40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.     

Identification and characterization of a serine protease from wheat leaves

A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of M_r = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca²⁺ and Zn²⁺, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses.     

Feeding fodder beet (Beta vulgaris L.) with either barley straw or pasture silage to non-lactating dairy cows

AIMS: To determine the suitability of diets containing either approximately 85% fodder beet (Beta vulgaris L.) with barley straw or 65% fodder beet with pasture silage when fed to non-lactating dairy cows, by measuring intakes, digestibility, rumen function including microbial growth, and N excretion.

METHODS: Holstein-Friesian cows fitted with permanent rumen fistulae were fed either 65% fodder beet with pasture silage (Silage; n=8) or 85% fodder beet with barley (Hordeum vulgare L.) straw (Straw; n=8) in an indoor facility over a 9-day period, for measurement of intakes, digestibility, rumen function and urine production. The cows were adapted to the diets over 2 weeks before the indoor measurements. Feed was available for about 6 hours/day, as practiced commercially for wintering non-lactating cows.

RESULTS: Five cows fed the Straw diet had to be removed from the trial because of acute acidosis; four on Day 1 of the measurement period and one on Day 7. One cow allocated to the Silage diet refused to eat fodder beet bulbs and was also removed from the trial. Two cows fed the Silage diet were also treated for acidosis. DM intakes were lower with the Straw than Silage diets (6.4 (SE 0.4) vs. 8.3 (SE 0.5) kg/day) and organic matter (OM) digestibility was lower with the Straw than Silage diets (77 (SE 1) vs. 83 (SE 1) g/100g). The N content of the two diets was 1.14 and 1.75?g/100?g DM and there was a net loss of N by cows fed the Straw diet (?22.7 (SE 7) g/day). Rumen microbial N production was much lower in cows fed the Straw than the Silage diet (6.6 (SE 1.3) vs. 15.8 (SE 0.7) g microbial N/kg digestible OM intake). Concentrations of ammonia in rumen liquid collected on Days 5–6 were below detection limits (<0.1?mmol/L) in 36/48 (75%) samples collected from cows fed the Straw diet and in 27/48 (56%) cows fed the Silage diet. Mean urinary N excretion was lower in cows fed the Straw than the Silage diet (52.0 (SE 5.8) vs. 87.7 (SE 5.9) g/day).

CONCLUSION AND CLINICAL RELEVENCE: An over-wintering diet for dry cows comprising about 65% fodder beet with 35% pasture silage provided adequate nutrition, although there was some risk of acidosis. In contrast, the diet containing about 85% fodder beet with barley straw resulted in lower DM intakes, poor rumen function, negative N balance so that both nutrition and welfare were compromised.     

Sanitary Procedures for the Production of Extended Semen

Semen is collected and processed from a variety of animal species for use in artificial insemination breeding programmes. Because of the inherent nature of the semen collection process, bacterial contamination of the ejaculate is a common occurrence. Additionally, manipulation of the ejaculate during processing in the laboratory can expose the sample to possible introduction of bacterial contamination. If preventative measures at the stud fail to adequately control these risks, decreases in semen quality, dose longevity and fertility may occur. Multiple mammalian and non-mammalian sources have been identified as origins of contamination in the stud. Knowledge of these sources has aided the industries in developing strategies that help in controlling the introduction of contaminant bacteria in extended semen. A primary step in minimizing contamination is in the practice of good hygiene by stud personnel. Prudent general sanitation protocols should also be followed in the laboratory, animal housing and semen collection areas. Cleanliness and attention to the actual semen collection process can also aid in reducing bacterial load originating from the stud semen donor. Attentiveness to all of these steps significantly contributes to an overall reduction in the type and amount of bacterial contamination. However, their complete elimination stills remains unavoidable. To address residual bacteria load in the sample, antimicrobials are commonly used in semen extenders intended to promote in vitro sperm longevity beyond that of a few hours. Current research by the animal industries continues in the selection and prudent use of antimicrobials that will lead to the success and sustainability of this modality in controlling bacterial contamination.