Gianfranco Gabai

Mammary apoptosis and lactation persistency in dairy animals.

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissues ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

The (193-209) 17-residues peptide of bovine β-casein is transported through Caco-2 monolayer.

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β-casein (β-CN, 193-209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β-CN (193-209) peptide across Caco-2 cell monolayer. In addition, we evaluated the possible routes of the β-CN (193-209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β-CN (193-209) peptide resisted the action of brush-border membrane peptidases, and that it was transported through the Caco-2 cell monolayer. The main route involved in transepithelial transport of the β-CN (193-209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. Our results demonstrated the transport of an intact long-chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.

Relationship between late embryonic mortality and the increase in plasma advanced oxidised protein products (AOPP) in dairy cows

The involvement of protein oxidation in embryonic mortality (EM) has been poorly investigated in cows. Advanced oxidation protein products (AOPP) are markers of protein oxidation generated by activated neutrophils and involved in inflammation. The aim of this work was to study AOPP in cow plasma and their relationship with late EM. The outcomes of 158 artificial inseminations (AI) were examined in 72 cows, which were classified ex post on the basis of blood progesterone and pregnancy-associated glycoprotein concentrations and clinical confirmation of pregnancy into the following categories: (1) positive (AI+, resulted in pregnancy, n=58), (2) negative (AI-, did not result in pregnancy, n=86) and (3) embryonic mortality (EM, n=14). Plasma protein fractions, malondialdehyde (MDA), total glutathione and AOPP were measured at AI (Day 0) and on Days 15, 28, 35, 45 and 60. MDA was significantly higher in EM than AI+ and AI- animals on Day 45, and than AI+ animals on Day 60 (P<0.05). Mean plasma AOPP concentrations were significantly higher in the EM group (P<0.01) and the ratio of AOPP:albumin was significantly higher in the EM group on Days 15, 28, 45 and 60 (P<0.05). Based on the temporal pattern of the AOPP:albumin ratio, we propose that oxidative stress is implicated in and may possibly be a cause of EM.

Oxidant/Antioxidant Balance in Animal Nutrition and Health: The Role of Protein Oxidation

This review examines the role that oxidative stress (OS), and protein oxidation in particular, plays in nutrition, metabolism, and health of farm animals. The route by which redox homeostasis is involved in some important physiological functions and the implications of the impairment of oxidative status on animal health and diseases is also examined. Proteins have various and, at the same time, unique biological functions and their oxidation can result in structural changes and various functional modifications. Protein oxidation seems to be involved in pathological conditions, such as respiratory diseases and parasitic infection; however, some studies also suggest that protein oxidation plays a crucial role in the regulation of important physiological functions, such as reproduction, nutrition, metabolism, lactation, gut health, and neonatal physiology. As the characterization of the mechanisms by which OS may influence metabolism and health is attracting considerable scientific interest, the aim of this review is to present veterinary scientists and clinicians with various aspects of oxidative damage to proteins.

Measuring fecal progestogens as a tool to monitor reproductive activity in captive female bottlenose dolphins (Tursiops truncatus)

The objective was to develop and test radioimmunoassays (RIAs) to measure fecal progestogens (P) and estrogens (E) to monitor ovarian activity in the bottlenose dolphin (Tursiops truncatus). Fecal samples were collected at least once a week for 20 mo from three peripubertal female bottlenose dolphins. Blood samples were collected at least once a month to compare serum and fecal steroid concentrations. Moreover, random fecal samples from three pregnant females, one lactating female, and one sexually mature female receiving oral altrenogest treatment were also collected. Fecal samples were collected behaviorally with a probe to avoid water contamination and extracted with petroleum ether (for P analysis) or diethyl ether (for E analysis). When possible, vaginal cytology and ovarian ultrasonography were used to monitor the estrous cycle. The RIA for fecal P had good reproducibility and negligible matrix effect. In addition, when fecal samples (N=25) were extracted with ethanol, the results with the two methods of extraction were highly correlated (r=0.923). Therefore, extraction of fecal samples with petroleum ether represented a valid alternative to other, more time-consuming methods of determining fecal P concentrations. In the absence of luteal activity, fecal P concentrations were consistently < 10 pmol/g feces, although they never decreased below 10 pmol/g during pregnancy. Thus, the threshold to confirm the presence of an active corpus luteum was provisionally set at 10 pmol/g. Around the onset of puberty, luteal phases appeared shorter and irregular in the bottlenose dolphin, as in other mammalian species. Additional HPLC-MS studies should be performed to identify predominant P metabolites to be used as fecal indicators of luteal activity in this species.

Usefulness of Nutraceutics in Controlling Oxidative Stress in Dairy Cows Around Parturition

Stress often results in the increased production of free radicals in organisms. Recent studies have indicated an interesting relationship between the presence of reactive oxigen species (ROS) and specific cellular activities in the mammary gland of lactating sheep (Colitti et al., 2002), and immunological activity (Kimura et al., 1999) and the ovarian cycle in dairy cows (Trout et al., 1998). An alternative strategy in the control of ROS generation, apart from vitamin E and C administration, is the inclusion in the diet of active compounds such as carotenoids and polyphenols (Jewell and O’Brien, 1999). The role and the activity of natural antioxidants not commonly present in the diets of ruminants (carotenoids and polyphenols of fruits) in the control of ROS generation is an interesting topic to investigate. The aim of this study was to evaluate the effects of dietary administration of nutraceutics with antioxidant properties, in the regulation of cellular redox homeostasis in ruminants.

Accuracy in naturally occurring anabolic steroid assays in cattle and first approach to quality control in Italy

This study assessed the accuracy of 16 commercial and three self-produced kits and drew the basis for using an external quality control (EQC) system. The commercial kits were mainly developed for blood sex steroid determination in humans but also have been used in cattle. Parallelism, recovery and precision tests were performed for progesterone (P4), testosterone (T) and oestradiol (E2) assays. Moreover, anonymous QC samples were sent to be analysed to some Italian laboratories. All kits showed a fair degree of parallelism (P < 0.01), even though 2/7 kits for T and 1/6 kits for E2 determination showed a regression coefficient (r2) lower than 0.98. For P4, an acceptable range of accuracy was achieved in the recovery test only by 1/6 kits; two kits showed fair or great overestimation and two kits considerable underestimation. For T, an acceptable range of accuracy was achieved only by 1/7 kits. For E2, 4/6 kits presented a variable degree of underestimation and two kits showed great overestimation. In the intra-assay precision test quite good repeatability was achieved only using samples with high hormone concentrations. While assaying samples with low concentrations we found a number of RSD > 10%. Moreover, the laboratories participating in the EQC produced statistically different (P < 0.05) results, particularly for high and medium concentrations. In conclusion, the use of commercial kits for screening naturally occurring sex steroid concentrations in cattle blood, in the case of suspected illegal treatments, requires preventive validation procedures and the development of an opportune EQC system.

Dehydroepiandrosterone secretion in dairy cattle is episodic and unaffected by ACTH stimulation

This paper describes the episodic release and response to adrenal stimulation of cortisol and dehydroepiandrosterone (DHEA) in cows. Observations made in samples taken every 10 min for 8 h (experiment 1) showed that plasma DHEA was significantly greater (P < 0.001) than DHEA-S, and release of these steroids was episodic and variable between animals (P < 0.01). No relationship was found between DHEA and cortisol. Significant (P < 0.001) DHEA-sulphate (DHEA-S) versus cortisol (R = -0.264) and DHEA-S versus DHEA (R = 0.200) correlations were found. DHEA and DHEA-S were not affected by a single ACTH challenge (experiment 2). In experiment 3, cortisol and DHEA secretions in response to prolonged ACTH administration (every 12 h for 6 days) were studied. On day 7, the episodic cortisol and DHEA release and response to the opioid antagonist naloxone were studied in blood samples taken every 10 min for 8 h. Animals were injected with naloxone after 4 h. A significant increase (P < 0.05) in mean circulating DHEA and DHEA pulse amplitude was observed during frequent sampling following ACTH treatment. DHEA and DHEA-S plasma concentrations were not affected following luteal regression (experiment 4). The effect of milk secretion around parturition on DHEA secretion was studied in dry and continuously milked cows (experiment 5). Plasma DHEA was significantly lower (P < 0.05) in milked cows. In the cow, ACTH is not an important DHEA secretagogue. Adrenal contribution to plasma DHEA is scarce. Likely, the placenta is the most important source of DHEA, and the lactating mammary gland can affect circulating DHEA levels. Investigation about the DHEA biological role in cows should be focused around parturition.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Protein corona as a proteome fingerprint: The example of hidden biomarkers for cow mastitis.

Proteome modifications in a biological fluid can potentially indicate the occurrence of pathologies, even if the identification of a proteome fingerprint correlated to a specific disease represents a very difficult task. When a nanomaterial is introduced into a biological fluid, macromolecules compete to form a protein corona on the nanoparticle surface, and depending on the specific proteome, different patterns of proteins will form the final protein corona shell depending on their affinity for the nanoparticle surface. Novel surface active maghemite nanoparticles (SAMNs) display a remarkable selectivity toward protein corona formation, and they are able to concentrate proteins and peptides presenting high affinities for their surface even if they are present in very low amounts. Thus, SAMNs may confer visibility to hidden biomarkers correlated to the occurrence of a pathology. In the present report, SAMNs were introduced into milk samples from healthy cows and from animals affected by mastitis, and the selectively bound protein corona shell was easily analyzed and quantified by gel electrophoresis and characterized by mass spectrometry. Upon incubation in mastitic milk, SAMNs were able to selectively bind αs2-casein fragments containing the FALPQYLK sequence, as part of the larger casocidin-1 peptide with strong antibacterial activity, which were not present in healthy samples. Thus, SAMNs can be used as a future candidate for the rapid diagnosis of mastitis in bovine milk. The present report proposes protein competition for SAMN protein corona formation as a means of mirroring proteome modifications. Thus, the selected protein shell on the nanoparticles results in a fingerprint of the specific pathology.