Analytical and clinical performance of a fluorescence enzyme immunoassay for progesterone and determination of ovulation day in bitches.

We evaluated the performance of a third-generation fluorescence enzyme immunoassay kit (FEIA; Tosoh Bioscience) for progesterone (P4) measurement in canine serum to identify the day of luteinizing hormone (LH) peak and ovulation in bitches. We conducted P4 assays on 54 serum samples using a FEIA and a chemiluminescence immunoassay (CLIA; Siemens). For the FEIA kit, the linearity test, recovery test, inter- and intra-assay CVs, and total error observed (TEo) were calculated. Serum samples from 28 bitches were used to evaluate the association of P4 concentration with the day of LH peak and the day of ovulation based on P4 thresholds (P4 ≥ 2 ng/mL, and doubling at 4-8 ng/mL in the following 48 h), and with pregnancy length. Linearity was 75-97% and 86-94% for high (37.0 ng/mL) and medium (3.8 ng/mL) pool serum samples, respectively. Recovery was 86.4-119%. Intra-assay CVs were 2.6%, 3.3%, and 5.2% for low (0.23 ng/mL), medium (6.24 ng/mL), and high (38.3 ng/mL) pool concentrations. Inter-assay CVs were 2.22% and 2.53% for P4 concentrations of 2.70 ng/mL and 8.2 ng/mL, respectively. TEo was 8.72% and 22.7% for P4 concentrations of 2 and 8 ng/mL, respectively. The mean pregnancy length from the day of LH peak and ovulation were 64.6 ± 1.7 and 63.3 ± 1.1 d, respectively. The third-generation FEIA kit that we tested was highly reliable.

Length of efficacy and effect of implant location in adult tom cats treated with a 9.4 mg deslorelin subcutaneous implant.

OBJECTIVES: The objective of this study was to assess duration of efficacy, side effects and return to fertility following use of the 9.4 mg deslorelin implant (Suprelorin 12; Virbac) in cats, and test whether efficacy and duration of action are influenced by implantation site (interscapular vs periumbilical). METHODS: Sixteen healthy adult tom cats were checked with (1) reproductive examination, (2) gonadotropin-releasing hormone stimulation test and (3) semen collection until achievement of sterility, then with (1) and (2) only at 2, 4, 6 and 12 months, and every 6 months thereafter until treatment effect disappeared. RESULTS: Serum testosterone reached basal levels by 7 days post-treatment. Semen quality improved initially then started to worsen by 1 month post-treatment and after 70 days post-treatment all cats were sterile. Early in the third month post-treatment there was a significant decrease in testicular volume and penile spikes. Testicular histology was normal upon neutering performed after resumption of fertility. No injection site lesions or treatment-related side effects were observed. There was no difference between periumbilical and interscapular placement for all criteria, but there was a trend for the decrease in testicular volume to last longer and for the regression of penile spikes to start sooner after interscapular administration. One of 16 cats did not respond to treatment. Six cats were lost at variable times during the study while fully responding to treatment. In the cats that completed the study, normal fertility was regained after 805 days, on average, but with a variable duration of effect from 750-850 days. CONCLUSIONS AND RELEVANCE: Treatment with a 9.4 mg deslorelin implant in male cats was effective for a period of 750-850 days, which is 1.5-2 times longer than the effect of the 4.7 mg deslorelin implant. Fertility (based on serum testosterone production and the presence of penile spikes) was regained at the end of the study. Placing implants in the intrascapular vs periumbilical location did not affect duration of suppression of testosterone production. The interscapular location may be characterised by a better efficacy, although further studies are needed to clarify this issue.

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.

CD49f+ mammary epithelial cells decrease in milk from dairy cows stressed by overstocking during the dry period.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

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.

Peripheral leukocyte populations and oxidative stress biomarkers in aged dogs showing impaired cognitive abilities.

In the present study, the peripheral blood leukocyte phenotypes, lymphocyte subset populations, and oxidative stress parameters were studied in cognitively characterized adult and aged dogs, in order to assess possible relationships between age, cognitive decline, and the immune status. Adult (N = 16, 2-7 years old) and aged (N = 29, older than 8 years) dogs underwent two testing procedures, for the assessment of spatial reversal learning and selective social attention abilities, which were shown to be sensitive to aging in pet dogs. Based on age and performance in cognitive testing, dogs were classified as adult not cognitively impaired (ADNI, N = 12), aged not cognitively impaired (AGNI, N = 19) and aged cognitively impaired (AGCI, N = 10). Immunological and oxidative stress parameters were compared across groups with the Kruskal-Wallis test. AGCI dogs displayed lower absolute CD4 cell count (p < 0.05) than ADNI and higher monocyte absolute count and percentage (p < 0.05) than AGNI whereas these parameters were not different between AGNI and ADNI. AGNI dogs had higher CD8 cell percentage than ADNI (p < 0.05). Both AGNI and AGCI dogs showed lower CD4/CD8 and CD21 count and percentage and higher neutrophil/lymphocyte and CD3/CD21 ratios (p < 0.05). None of the oxidative parameters showed any statistically significant difference among groups. These observations suggest that alterations in peripheral leukocyte populations may reflect age-related changes occurring within the central nervous system and disclose interesting perspectives for the dog as a model for studying the functional relationship between the nervous and immune systems during aging.

Relationship between plasma progesterone and pregnancy-associated glycoprotein concentrations during early pregnancy in dairy cows.

The relationship between the concentration of plasma progesterone (P4) during embryo attachment or at recognition of pregnancy, and that of pregnancy-associated glycoprotein (PAG) was assessed in dairy cows. The outcome of artificial insemination (AI) was classified as positive (AI+), negative (AI-), or late embryonic mortality (EM) by measuring circulating PAG concentrations and by ultrasonography. Based on P4 concentrations at either day 21 or day 15, AI+ and EM cows were classified into 'low' (P4 concentrationsmean) P4 groups. In both experiments, the threshold of P4 concentration between the 'low' and 'high' groups was approximately 6ng/mL. PAG concentrations were lower in the 'low' group only when P4 concentrations were below the threshold. The study findings suggest that a possible P4 threshold exists below which PAG secretion may be impaired.

Oxidative stress indicators and metabolic adaptations in response to the omission of the dry period in dairy cows.

The effects of dry period omission on oxidative stress and metabolic indicators around calving were studied. Seventeen Italian Friesian cows were randomly assigned to two groups, homogeneous for milk yield and parity, and managed either with a traditional 55-d dry off period (n=8) or continuously milked till parturition (n=9). Between 60 d before expected calving and 90 d after calving, body condition (BCS) was recorded and blood samples were collected to measure cortisol, urea, cholesterol, glucose, NEFA, triglycerides, insulin, malondialdehyde (MDA), total glutathione (GSH) and glutathione peroxidase (GPx) activity. BCS changes after calving were not different between the two groups. The normally dried group showed lower (P<0.05) glucose concentrations on day 7 before calving, greater (P<0.01) non-esterified fatty acid concentrations at 7 d and 15 d after calving, and greater (P<0.01) triglyceride concentrations for all the period before calving. On the other hand, plasma MDA was not different between groups. On average, plasma GSH concentrations were greater in continuously milked cows after calving (P<0.05), while plasma GPx was greater with continuous milking up to parturition (P<0.01). The results confirmed that omitting the dry period leads to an improved energy balance. The degree of oxidative stress was not detrimental for animal health, and the slight modifications of GPx observed prepartum were possibly related to continuous milk secretion. The differences in plasma GSH observed after calving may depend upon sulphur amino acid sparing in continuously milked cows.

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.

Species-specific chemosignals evoke delayed excitation of the vomeronasal amygdala in freely-moving female rats.

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species-specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species-specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO-mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species-specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.

Mammary steroid metabolizing enzymes in relation to hyperplasia and tumorigenesis in the dog.

Progesterone and estradiol play a crucial role in the control of mammary gland proliferation and tumour formation in the dog. However, little is known whether steroid metabolizing enzymes are present within the canine mammary gland that may play a modulating role in the bioavailability of progesterone and estrogen. In this study we investigated the expression of the steroid metabolizing enzymes 5alpha-reductase (type I and type II) and aromatase in relation to hyperplasia or tumorigenesis in the canine mammary tissue. The relative mRNA concentrations were examined by a semi-quantitative reverse-transcriptase PCR analysis (RT-PCR). In addition the affinity of dihydroprogesterone (5alpha-reduced metabolite of progesterone) for canine progesterone receptors was investigated. Quantification of the RT-PCR products revealed that in mammary tumours a significantly higher expression of aromatase is present in comparison to normal mammary tissue. Furthermore, significant decrease in expression of both aromatase and 5alpha-reductase type II enzymes was found in hyperplasic mammary tissue compared to tumours. The changes in expression of type II 5alpha-reductase and aromatase were highly correlated. 5alpha-Reduction of progesterone to dihydroprogesterone resulted in a six-fold less affinity for the canine progesterone receptor. It is concluded that hyperplasia is associated with a decreased expression of type II 5alpha-reductase and aromatase enzymes, whereas in tumours the opposite situation is found.

The increase in plasma C19Delta5 steroids in subcutaneous abdominal and jugular veins of dairy cattle during pregnancy is unrelated to estrogenic activity.

Plasma concentrations of dehydroepiandrosterone (DHEA), 5-androstene-3beta,17beta-diol (AED), and 17beta-estradiol (E2) in dairy cows and heifers and AED binding to uterine cytosolic estrogen receptor (ER) were studied. Plasma samples were collected from the subcutaneous abdominal (SA) and jugular (J) veins of heifers and cows in the non-pregnant state and at 15-45, 90-120, 180-210, and 250-280 days of pregnancy (N = 5-12). Plasma DHEA, AED, and E2 were determined by RIA. DHEA and AED significantly increased (P < 0.001) in heifers and cows throughout pregnancy. The stage of pregnancy significantly (P < 0.001) affected the three steroids in heifers and cows. Plasma DHEA increased throughout pregnancy in both heifers and cows, and in heifers it was significantly greater in SA than in J veins at 90-120 days (P < 0.01). Plasma AED was greater in heifers than in cows in J veins at 90-120 days (P < 0.01) and 180-210 days (P < 0.05), and in SA veins, at 15-45 days (P < 0.01) and 90-120 days (P < 0.05). In heifers, circulating AED showed concentration values significantly greater than those in non-pregnant animals from 90 to 120 days (P < 0.05) and was significantly greater in SA than in J veins at 90-120 days (P < 0.05). In cows, plasma AED was significantly greater than in non-pregnant animals at 250-280 days (P < 0.01). In heifers, plasma E2 was significantly greater in the SA than in the J veins from 180-210 to 250-280 days (P < 0.01). In cows, differences between E2 plasma concentrations in J and SA veins were observed only at 250-280 days of pregnancy. At 250-280 days, in both animal types plasma E2 was significantly greater than in non-pregnant animals (P < 0.001). We suggest that AED originates primarily from the feto-placental unit, while mammary E2 synthesis near term can affect plasma concentrations. Binding data showed that AED is a weak competitor for cytosolic ER (IC50 range: 1.44 x 10(-5) to 3.71 x 10(-5) M). These results suggest that a direct estrogenic activity for AED is unlikely in dairy cattle, and the physiological role of AED needs to be elucidated.

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 tissue's 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.