Temperature and humidity index (THI) affects salivary cortisol (HC) and dehydroepiandrosterone (DHEA) concentrations in growing bulls following stress generated by performance test procedures.

The hypothalamus-pituitary-adrenal axis response to a challenge was proposed for genetic selection of robust and resilient animals. As ACTH (adrenocorticotropic hormone) test and hormone measurements in blood may result impractical, it may be useful to measure salivary hormones in response to natural stressors, after an accurate biological validation, to control factors that could contribute to the response. We evaluated whether animal handling during performance test affects salivary HC and DHEA secretion and could be used for selection. We tested the effects of habituation to repeated handling and THI as putative bias. Bull calves (N = 273) undergoing performance test were sampled at 8-9 and 11-13 months (N = 101), 8-9 months (N = 131), or 11-13 months (N = 41). On each test day (D0), calves were isolated, conducted to a squeeze chute and immobilized for 6 min. Saliva samples were collected in the morning after feed administration (T0), and after 6 min immobilization in the squeeze chute (T1) for HC and DHEA measurement. Environmental temperature and relative humidity were recorded every hour from 1:00 h to 24:00 h during the 6 days before the performance test and on D0. Salivary HC and DHEA concentrations were higher in T1 (p < 0.01), although a clear individual positive response to handling could be observed in less than 10% of subjects. The mixed model revealed: (i) HC and HC/DHEA were higher in Young bulls (p < 0.05). (ii) The time of T0 sample collection significantly affected DHEA (p < 0.01) and HC/DHEA (p < 0.05). (iii) THI affected both steroids (p < 0.001) but not HC/DHEA. Spearman correlations suggested that THI weakly affected salivary HC at T0 only (ρ = 0.150, p < 0.01), while moderate statistically significant correlations were found between DHEA and THI at T0 (ρ = 0.316, p < 0.001), and T1 (ρ = 0.353, p < 0.001). Salivary HC and DHEA in response to handling procedures might identify subpopulations of subjects with sensitive HPA axis. Habituation to repeated handling played a role, as the hormone response was lower in older animals. Chronic exposure to high THI had a minor effect on salivary HC visible at T0. A more intense THI effect was observed on salivary DHEA concentrations at both T0 and T1, which should be worth of further investigations.

Relationships between Biomarkers of Oxidative Stress in Seminal Plasma and Sperm Motility in Bulls before and after Cryopreservation.

This study aimed at evaluating the relationship between biomarkers of oxidative stress (OS) in seminal plasma and sperm motility in bulls before and after cryopreservation. Three ejaculates per bull were collected from 20 young bulls. Each ejaculate was analyzed for motility before and after cryopreservation (by CASA), and the SP concentration of Advanced Oxidation Protein Products (AOPP), thiols, and carbonyl groups (CT) were examined. Then, based on their motility, the ejaculates were grouped into: high motility fresh (HMF), low motility fresh (LMF), high motility thawed (HMT), and low motility thawed (LMT) groups. Higher AOPP and thiol concentrations on SP were related (p < 0.05) to the higher LIN and BCF and lower ALH of fresh semen. In addition, AOPP and thiols were significantly higher in HMF than LMF. As a confirmation of this, the Receiver Operating Characteristic (ROC) curve analysis showed that AOPP and thiol concentrations in SP were able to discriminate between HMF and LMF ejaculates (Area Under the Curve of 71.67% and 72.04%, respectively). These observations give an alternative perspective on the relationship between sperm motility and the OS parameters of SP, which need further investigations.

Case Report: Repeated Intralesional Injections of Autologous Mesenchymal Stem Cells Combined With Platelet-Rich Plasma for Superficial Digital Flexor Tendon Healing in a Show Jumping Horse.

In the present case report a show jumping 10-year-old Sella Italiano gelding, presented with severe lameness, swelling and pain at palpation of the mid-metacarpal region of the left forelimb. Clinical and ultrasound examination diagnosed a chronic tendonitis of the central region of the superficial digital flexor tendon (SDFT). The lesion was a reoccurrence since it developed from a previously healed injury. The horse had to stop competing and was unresponsive to gold-standard treatments as Non-steroidal anti-inflammatory drugs (NSAIDs) and conservative management after 6 months of therapy. The animal was subjected to repeated intralesional injections of autologous adipose-derived mesenchymal stem cells (AD-MSCs) combined with autologous platelet-rich plasma (PRP). The combined treatment was administered twice in a 1-month interval. The healing process was assessed through clinical examination, ultrasound imaging and quantification of oxidative stress products and inflammatory mediators in blood plasma. After 2 weeks from first injection, a reduction of concentration of oxidative-derived products was observed, together with an increase of anti-inflammatory cytokines and pro-mitotic growth factors. These results were reflected clinically as the horse showed a reduction of lameness along with swelling and pain after 4 weeks. At the 1-year follow-up, the horse showed no signs of lameness and swelling. The ultrasonographic examination highlighted a compact fiber alignment with a normal echogenic tendon as observed in the sound contralateral limb. Moreover, the horse went back to the previous level of competition. Our results suggest the positive effects of a repeated intralesional injection of AD-MSCs and PRP for the treatment of a chronic tendonitis with long-term effects and an improvement for both equine quality of life and athletic performance.

Repeated intra-articular administration of equine allogeneic peripheral blood-derived mesenchymal stem cells does not induce a cellular and humoral immune response in horses.

OBJECTIVE: The use of mesenchymal stem cells (MSCs) for the treatment of equine joint disease is widely investigated because of their regenerative and immunomodulatory potential. Allogeneic MSCs provide a promising alternative to autologous MSCs, since the former are immediately available and enable a thorough donor screening. However, questions have been raised concerning the immunogenic potential of allogeneic MSCs, especially after repeated administration. METHODS: Current retrospective study assessed the cellular and humoral immunogenicity of ten jumping and dressage horses with naturally occurring degenerative joint disease which were treated 3 times intra-articularly with a 1 mL stem cell suspension containing 1.4-2.5 million chondrogenic induced equine allogeneic peripheral blood-derived MSCs (ciMSCs) combined with 1 mL equine allogeneic plasma. Stem cells from 2 donor horses were used. Horses were clinically evaluated for joint effusion, presence of pain to palpation and skin surface temperature at the local injection site, joint range of motion, occurrence of adverse events and the presence of ectopic tissue. The cellular immune response was analyzed using a modified mixed lymphocyte reaction and the humoral immune response was investigated using a flow cytometric crossmatch assay by which the presence of alloantibodies against the ciMSCs was evaluated. Presence of anti-bovine serum albumin antibodies was detected via ELISA. RESULTS: Clinical evaluation of the horses revealed no serious adverse effects or suspected adverse drug reactions and no ectopic tissue formation at the local injection site or in other areas of the body. Generally, repeated administration led to a decrease of horses with joint effusion of the affected joint. Pain to palpation, skin surface temperature and joint range of motion did not increase or even decreased after treatment administration. Allogeneic ciMSCs did not induce a cellular immune response and no alloantibodies were detected in the recipients' serum, regardless the presence of BSA antibodies in 70 % of the horses. CONCLUSION: Repeated intra-articular injections with allogeneic equine ciMSCs did not elicit clinically relevant adverse events. Furthermore, current study indicates the absence of a cellular or a humoral immune response following repeated intra-articular injections.

Cellular and Humoral Immunogenicity Investigation of Single and Repeated Allogeneic Tenogenic Primed Mesenchymal Stem Cell Treatments in Horses Suffering From Tendon Injuries.

The use of mesenchymal stem cells (MSCs) for the treatment of equine tendon disease is widely investigated because of their regenerative and immunomodulatory potential. However, questions have been raised concerning the immunogenic properties of allogeneic MSCs. Therefore, two studies were conducted to assess the safety of equine allogeneic peripheral blood-derived tenogenic primed MSCs (tpMSCs). The objective was to evaluate if a single and repeated tpMSC administration induced a cellular and humoral immune response in horses suffering from tendon injuries. Horses enrolled in the first study (n = 8) had a surgically induced superficial digital flexor tendon core lesion and were treated intralesionally with tpMSCs. Before and after treatment the cellular immunogenicity was assessed by modified mixed lymphocyte reactions. The humoral immune response was investigated using a crossmatch assay. Presence of anti-bovine serum albumin (BSA) antibodies was detected via ELISA. Horses enrolled in the second study (n = 6) suffered from a naturally occurring tendon injury and were treated twice with tpMSCs. Blood was collected after the second treatment for the same immunological assays. No cellular immune response was found in any of the horses. One out of eight horses in the first study and none of the horses in the second study had anti-tpMSC antibodies. This particular horse had an equine sarcoid and further investigation revealed presence of antibodies against sarcoid cells and epithelial-like stem cells before treatment, which increased after treatment. Additionally, formation of antibodies against BSA was observed. These findings might indicate a non-specific immune response generated after treatment. Serum from the other horses revealed no such antibody formation. These two studies showed that the administration of tpMSCs did not induce a cellular or humoral immune response following an intralesional single or repeated (two consecutive) allogeneic tpMSC treatment in horses with tendon injury, except for one horse. Therefore, a larger field study should confirm these findings and support the safe use of tpMSCs as a therapeutic for horses suffering from tendon injuries.

Fatty Acid Profile and Enterolactone Content of Early and Commercial Milk of Dairy Cows Supplemented with Flaked Flaxseed during the Dry Period.

Various supplementations in animal feeding have been investigate in order to enrich food of animal origin with n-3 fatty acids. Although the effects of flaxseeds inclusion in diets for lactating dairy have already been assessed, few studies have focused on this n-3 source supplementation during the transition period. The aim of this work was to evaluate the effects of flacked flaxseed (200 g/head/day; 2.13% DM) dietary treatment during the dry period on milk yield and quality in the 30 days after calving. In addition, the enterolactone content in plasma (before and after calving) and in milk of cows fed diets supplemented or not with flaxseed was considered. The study demonstrated that the carry-over effect on the milk profile of C18:2, C18:3 n-3, and C20:5 n-3 was significantly higher in flaxseed diet than in the control one at 4th day of lactation. A significant increase of enterolactone on milk from flaxseed fed cows was observed only at 15 sampling day. The quick modification in fatty acid (FA) profile of the milk in the first few days of lactation suggests that the carry over effect from pre-calving flaxseed feeding at this concentration was very short lasting.

Oxidative status of erythrocytes, hyperglycemia, and hyperlipidemia in diabetic cats.

BACKGROUND: Erythrocytes of diabetic cats have decreased superoxide dismutase activity, possibly indicative of oxidative stress. HYPOTHESIS: Erythrocytes of diabetic cats undergo oxidative stress, which is caused by hyperglycemia and hyperlipidemia, and improves with treatment. ANIMALS: Twenty-seven client-owned cats with diabetes mellitus, 11 matched healthy cats, and 21 purpose-bred healthy cats. METHODS: Prospective study. Advanced oxidized protein products, carbonyls (protein oxidation by-products), and thiols (antioxidants) were quantified in erythrocyte membrane, thiobarbituric acid reactive substances (TBAR, lipid peroxidation by-products), and thiols in erythrocyte cytoplasm of all cats. Comparison were performed between diabetic and matched healthy cats, between diabetic cats achieving remission or not, and among purpose-bred cats after 10 days of hyperglycemia (n = 5) or hyperlipidemia (n = 6) versus controls treated with saline (n = 5) or untreated (n = 5). RESULTS: Compared with controls, erythrocytes of diabetic cats initially had higher median membrane carbonyls (4.6 nmol/mg total protein [range: 0.1-37.7] versus 0.7 [0.1-4.7], P < .001) and lower cytoplasmic TBAR (1.9 nmol/mg [0.5-2.4] versus 2.4 [1.4-3.5] P < .001), and thiols (419 nmol/mg [165-621] versus 633 [353-824], P < 0.001). After 12-16 weeks of treatment in diabetic cats, carbonyls decreased by 13% (P < .001), but remained higher (P < .001) and TBAR and thiols lower (P = .02, P < .001) than those in controls. No differences were observed between diabetic cats achieving remission or not, and among purpose-bred cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Diabetes mellitus is associated with increased protein oxidation and reduced antioxidant defenses, which persist during treatment and remission, although mild improvement in protein oxidation occurs. Short-term hyperglycemia or hyperlipidemia does not cause oxidative stress. The reason for decreased TBAR remains unknown.

Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans.

Immunology of marine mammals is a relatively understudied field and its monitoring plays an important role in the individual and group management of these animals, along with an increasing value as an environmental health indicator. This study was aimed at implementing the knowledge on the immune response in cetaceans stranded along the Italian coastline to provide a baseline useful for assessing the immune status of bottlenose (Tursiops truncatus) and striped (Stenella coeruleoalba) dolphins. In particular, since the Mediterranean Sea is considered a heavily polluted basin, a comparison with animals living in open waters such as the Atlantic Ocean was made. Formalin-fixed, paraffin-embedded spleen, thymus, and lymph node tissues from 16 animals stranded along Italian and 11 cetaceans from the Canary Island shores were sampled within 48 h from death. Information regarding stranding sites, gender, and age as well as virologic, microbiological, and parasitological investigations, and the cause and/or the death mechanism were also collected in order to carry out statistical analyses. Selected tissues were routinely stained with hematoxylin-eosin (H&E) and with immunohistochemical techniques (IHC). For IHC analysis, anti-human CD5 monoclonal mouse antibody to identify T lymphocytes, CD20 monoclonal mouse antibody for the identification of mature B lymphocytes and HLA-DR antigen (alpha-chain) monoclonal mouse antibody for the identification of the major histocompatibility complex type II were previously validated for both species by Western-blotting technique. T-test method applied to quantitative evaluation of IHC positive cells showed a significant relationship between the number of (expression) of CD20 stained lymphocytes and normal and hypoplastic lymph nodes, respectively. No other significant correlations were noticed. Analyses for organochlorines (OC) compounds were performed in animals (n°5) having frozen blubber tissue available. A simple linear regression was calculated to predict if the amount of OCs could influence the number of inflammatory cell subpopulations and a moderate negative correlation was found between the presence of high quantity of contaminants and the number of T lymphocytes. Future analysis should be aimed to understand the effect of the major immunomodulatory pathogens on sub-populations of B and T cells.

Relationship between Protein Oxidation Biomarkers and Uterine Health in Dairy Cows during the Postpartum Period.

High neutrophil (PMN, Polymorphonuclear neutrophil) counts in the endometrium of cows affected by endometritis, suggests the involvement of oxidative stress (OS) among the causes of impaired fertility. Protein oxidation, in particular, advanced oxidation protein products (AOPP), are OS biomarkers linked to PMN activity. To test this hypothesis, the relationship between protein oxidation and uterus health was studied in thirty-eight dairy cows during the puerperium. The animals were found to be cycling, without any signs of disease and pharmacological treatments. PMN count was performed either through a cytobrush or a uterine horn lavage (UHL). Cows were classified into four groups, based on the uterine ultrasonographic characteristics and the PMN percentage in the uterine horns with a higher percentage of high neutrophil horn (HNH). They were classified as: Healthy (H); Subclinical Endometritis (SCE); Grade 1 Endometritis (EM1); and Grade 2 Endometritis (EM2). AOPP and carbonyls were measured in plasma and UHL. UHL samples underwent Western blot analysis to visualize the carbonyl and dityrosine formation. Plasma AOPP were higher (p < 0.05) in EM2. AOPP and carbonyl group concentrations were higher in the HNH samples (p < 0.05). Protein concentration in the UHL was higher in the EM2 (p < 0.05). Carbonyl and dityrosine formation was more intense in EM1 and EM2. Protein oxidation observed in the EM2 suggests the presence of an inflammatory status in the uterus which, if not adequately hindered, could result in low fertility.

Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of aS1-casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.

One year daily changes in fecal sexual steroids of two captive female cheetahs (Acinonyx jubatus) in Italy.

The present study evaluated changes of fecal sexual steroids in two female cheetahs (Geijsha and Duchessa) in Northern Italy throughout one year. Wet feces were collected daily from two sibling animals of the same age, housed with conspecific males and managed in the same conditions, and estrogens and progestogens concentrations were analyzed by radioimmunoassay (RIA). Evidence of ovarian activity based on regular fluctuation in estrogen excretion was demonstrated in both females. None of the animals was continuously cycling, as follicular activity was interrupted by anestrous periods, during the spring and early winter. No significant increases of progestogens were recorded after the estrogen peaks, indicating that induced or spontaneous ovulations did not occur during the observation period. The wavelet decomposition evidenced the temporal pattern of ovarian activity in the two females, underlying throughout the year a more pronounced rhythmical ovarian estrogenic activity in Geijsha than in Duchessa. However, this statistical approach had a smoothing effect in depicting the hormonal patterns and the number of follicular phases might be lower than that revealed by the iterative method. In this study, RIA on wet feces performed very well to determine sexual steroid concentrations, and an ovarian activity interrupted by anestrous periods along the year in captive cheetahs co-housed in a small group was demonstrated. More information on estrous behavior of captive cheetahs were obtained in this study, but the effects of husbandry and management conditions on natural reproductive physiology of this species remain to elucidate.

Milk cortisol response to group relocation in lactating cows.

The aim of the study reported in this Research Communication was to analyse the variations of milk cortisol concentrations in response to the relocation of dairy cows between production groups. Milk cortisol measured during 3 consecutive days did not vary significantly in cows without environmental perturbation. However, relocation of cows caused a significant increase of cortisol in milk starting from the first milking after the group change. This suggests that cortisol in milk can be a suitable biomarker to assess the HPA response of dairy cows to a short/medium-term environmental challenge.

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.

Age and isolation influence steroids release and chemical signaling in male mice.

Social interactions in mice involve olfactory signals, which convey information about the emitter. In turn, the mouse social and physiological status may modify the release of chemical cues. In this study, the influences of age and social isolation on the endocrine response and the release of chemical signals were investigated in male CD1 mice, allocated into four groups: Young Isolated (from weaning till 60days; N=6), Adult Isolated (till 180days; N=6), Young Grouped (6 mice/cage; till 60days; N=18), Adult Grouped (6 mice/cage; till 180days; N=18). Mice were transferred in a clean cage to observe the micturition pattern and then sacrificed. Body and organs weights, serum testosterone, dehydroepiandrosterone, corticosterone and the ratio Major Urinary Protein/creatinine were measured. Urinary volatile molecules potentially involved in pheromonal communication were identified. Androgen secretion was greater in isolated mice (P<0.05), suggesting a greater reactivity of the Hypothalamic-Pituitary-Gonadal axis. Grouped mice presented a higher degree of adrenal activity, and young mice showed a higher serum corticosterone (P<0.05) suggesting a greater stimulation of the Hypothalamic-Pituitary-Adrenal axis. The micturition pattern typical of dominant male, consisting in voiding numerous droplets, was observed in Young Isolated mice only, which showed a higher protein/creatinine ratio (P<0.05). Urinary 2-s-butyl-thiazoline was higher in both Young and Adult Isolated mice (P<0.005). Young Isolated mice showed the most prominent difference in both micturition pattern and potentially active substance emission, while long term isolation resulted in a less extreme phenotype; therefore social isolation had a higher impact on young mice hormone and pheromone release.

Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro.

Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers. Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P<0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P<0.001). Activation with PMA induced a loss of viable neutrophils after 3h, which was greater with AOPP-BSA incubation (P<0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.

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.

Factors affecting progesterone production in corpora lutea from pregnant and diestrous bitches.

Factors affecting the characteristics of corpora lutea (number, left/right ovary origin, weight, DNA and progesterone content) were studied in 73 healthy bitches divided into two classes of age (< or =2.5 vs. >2.5 years; mean+/-S.E.=3.6+/-0.3 years; range: 0.7-10 years), weight (< or =20 vs. >20 kg; mean+/-S.E.=16.2+/-1.2 kg; range: 5-45 kg), reproductive status (pregnancy vs. diestrous; pregnant bitches N=41 and diestrous bitches N=32), stage of luteal phase (20-40 vs. 41-55 days) and ovulation rate (< or =7 vs. >7). Two different assessments were performed: (a) comparison of luteal tissue characteristics and progesterone content between pregnant and diestrous bitches and (b) investigation of the effect of animal age, weight and ovulation rate on individual corpus luteum (CL) parameters. None of the luteal parameters differed between pregnant and diestrous bitches, even when the stage of the luteal phase was considered. Age and weight of the bitch significantly influenced luteal tissue characteristics: heavier bitches had more and heavier CLs (P<0.001) and carried more foetuses (P<0.01), while older bitches had a higher number of CLs (P<0.001). In pregnant animals, the rate of foetuses to Cls was 78.4%. Luteal progesterone content was significantly affected by the ovulation rate (P<0.01). A significant individual effect (P<0.0001) was present on all the parameters in the single CL, with the right ovary carrying a higher CL number (P<0.01), with greater DNA (P<0.01) and P4 content (P<0.001). CLs of younger bitches showed a diminished efficiency of P4 production (P4/mg, P4/DNA) with a significant effect of the interaction between age and reproductive condition of the bitch on DNA and progesterone content (P<0.0001). These findings indicate that animal weight and age have a major influence on the characteristics of canine corpora lutea.

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.