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This study investigated the effects of dietary linseed and physiological state on blood concentrations of some metabolic hormones and indicators, in transition dairy ewes. From 21 d before lambing to 60 d post-partum, ewes were provided with one of three isoenergetic and iso‑nitrogenous pelleted concentrates, without (CTR, n = 21), or with 100 (EL-10, n = 22) or 200 g/kg (EL-20, n = 22) of extruded linseed. Animals were fed alfalfa hay ad libitum and had access to mixed pasture. Plasma thyroid hormones and insulin concentrations were not affected by diet and significantly changed by time. The last week of pregnancy T3 (2.27 ± 0.15 ng/mL) and T4 (102.63 ± 2.85 ng/mL) were lower than at 4 weeks before lambing (T3: 3.27 ± 0.27 ng/mL; T4: 125.89 ± 2.63 ng/mL). After lambing, T3 rose to be highest at 4 and 8 weeks of lactation (3.41 ± 0.22). Mean insulin peaked 2 weeks after lambing (0.31 ± 0.02 ng/mL) vs. late pregnancy (0.22 ± 0.01 ng/mL) and progressing lactation (0.18 ± 0.01 ng/mL). Plasma Leptin concentration (2.43 ± 0.03 ng/mL) was not affected by diet nor time. All the blood metabolites and enzymes investigated showed significant time x treatment interaction. Differences of several haematological parameters were found in EL vs. CTR (cholesterol, triglycerides, total protein, bilirubin, ALP, ALT), however, in most cases the values were fairly within the reference physiological ranges. Metabolic hormones are confirmed to be mainly linked to the different physiological states, energy intake and variations of energy balance, without clear effects by different sources of energy and quality of dietary lipids.

The management of shelter dogs whose dangerousness to people has been verified is an aspect of considerable importance as it assesses animal welfare, public health, and the management of human and economic resources. In this paper, we briefly discuss the case of a large sized male dog that had bitten people several times and was declared to be at high risk of causing danger. Despite a behavioral rehabilitation program, the initial evidence of dangerousness remained unchanged, thus, there was no possibility of putting the dog up for adoption. This clinical case is an example of how conflicting it is for a behaviorist to choose ethically when considering euthanasia and animal welfare.

Simple Summary In Central Italy Apennine sheep represent the most bred species, which is still bred today in a semi-extensive manner, exploiting the natural pastures. However, the increase in summer aridity induces a decrease in the pastoral value of the grasslands, resulting in poor animal performance and production. Only research-derived innovation can support farmers’ economy in order to maintain the pastoral activities. The objective of this study was to evaluate the effects of cereals supplementation on body condition score and metabolic hormones profile in milking ewes grazing on semi-natural pastures on mid mountain rangelands. Our findings showed that feed supplementation preserves grazing ewes from the usual lowering of the body state associated to the lactation period and sustains the metabolic status of animals. Taken together, our results could represent a helpful instrument in the farm management practices. Abstract This study aimed to investigate the effects of feed supplementation on body condition score (BCS) and different metabolic hormones concentration in lactating sheep reared in Italian Central Apennine pastures during the grazing summer period. In this study, 24 Comisana x Appenninica pluriparous ewes from June until August were divided into two homogenous groups: the control group (UNS) was free to graze, while the other group (SUP), in addition to grazing, was supplemented with 600 g/day/head of cereals. At the start of the supplementation and at an interval of 9–10 days until the end of experimentation, BCS evaluation and blood withdrawal to assay nesfatin-1, insulin, glucagon, leptin, triiodothyronine and cortisol levels were performed. Univariable analysis showed no remarkable differences between the groups, while multivariable analysis suggested that the UNS group was characterized by a lower BCS and greater nesfatin-1 than the SUP group. These findings can be considered in relation to milk production, which shows a clear better persistence in the SUP group. Our results indicate that nutritional supplementation has protected ewes from the usual lowering of the body state linked to lactation and provides a good maintenance of milk production, determining also a better overall body and metabolic state of the animals, which is important at the beginning of the sexual season.

An instance of hormone assay method flaw is reported. In this journal Chronobiology International, two papers appeared in which an ELISA method for human serum or plasma was utilized for blood serum of horse and sheep, respectively. From our testing, it is resulted that such method does not work at all for equine, sheep and other animal species. The use of commercial hormone assay kits for heterologous species always needs a careful validation procedure. First, the same hormone molecule by different species could not share enough homology to be regognized by and react with antibodies utilized in the method. Furthermore, even with a full overlap of the molecules, possible interferences by other components of the sample (matrix effect) have to be considered.

Grazing is a main tool for biodiversity conservation, however, there is a risk of pasture abandonment due to the climate changes leading to an increasing summer aridity that negatively affect forage features; worsening in forage composition resulted in decline of sheep body status, mainly due to the rise of fiber content, which causes the increase of rumen keratinization degree. During summer, the deterioration of the body condition of sheep is the main cause of the declining milk yield and quality; this can have serious impacts on farm household income, enhancing the risk of pasture abandonment with consequent environmental and territorial degradation. This short note presents the food supplementation as an essential way to contrast the rapid rumen keratinization in sheep fed on the pasture during the summer. A flock of 45 adult female sheep were conducted on pasture at the beginning of June 2016, where they were free to grazing until the moment of pasture maximum flowering. From this moment until the maximum pasture dryness, the animals were divided in two groups: the control group fed only on the pasture, while the experimental group was also supplemented with 600 g/day/animal of corn and barley (1:1). The rumen epithelium keratinization degree, Body Condition Score and milk production were monitored at the beginning and at the end of the period of differentiated diets. The food supplementation did not affect the animal body state, but it slow down the growth of the rumen keratinization degree, allowing a better absorption of nourishing principles and then bringing to a significant increase of the milk production. An analysis of cost/benefit, attempted also considering the newborn numerousness, indicated that food supplementation could mitigate the productive loss induced by summer aridity preserving the economic sustainability of sheep milk production, thus avoiding land abandonment.

Aged dogs may develop age-related neuropathological and behavioral changes including the reduction in their memory and learning abilities. The aim of the study was to assess the effects of dietary supplementation with tramiprosate (homotaurine) on spatial cognition of aging dogs. Twenty-four aged dogs, sheltered in the same kennel, were randomly assigned into two groups—one group receiving homotaurine supplementation (500 mg once daily) and the other group, living in the same environment, without any supplementation. Cognitive testing was conducted using a T-maze. The experimental procedure consisted of four stages: a one-time direction-determination trial and a learning task, memory task, and reversal learning task that were done at T zero (immediately before supplementation start) and repeated after 4, 8, and 12 months. Data were collected from video-recordings by a single observer. The homotaurine-supplemented dogs were able to maintain a better performance in learning and memory trials with the largest differences observed in the reversal learning test, with a higher number of dogs able to pass the test (P

This is the first time that PAG determination using two different antisera raised against PAG molecules purified from both caprine (RIA‐706) and bubaline placentas (RIA‐860) is reported in water buffalo. Ninety‐eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) programme, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and 45. Pregnancy was confirmed by ultrasonography on days 28 and 45. The blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 postcalving. Differences in PAG concentrations were observed between pregnant and nonpregnant buffaloes starting from day 23 post AI using both RIA‐706 and RIA‐860 (p < 0.001). However, estimated mean concentrations of PAG measured by RIA‐706 were higher than RIA‐860 (p < 0.001) and Bland–Altman analysis showed biases ranged from 0.0 ng/ml at day 23 to 0.79 ng/ml at day 28 post AI. Moreover, RIA‐706 showed greater sensitivity and accuracy both at 23 and 25 days of pregnancy. RIA‐706 and RIA‐860 decreased below 1 ng/ml from 40 and 30 days postpartum, respectively, suggesting that PAG are better recognized by the antisera raised against the caprine PAG in the postpartum period also. This is essential when using PAG as an appropriate marker of early pregnancy after postpartum for detecting new pregnancies. The results of this study show that the ability of RIA systems to recognize early PAG could be improved using antisera raised against PAG molecules isolated from caprine placenta.

The aim of this study was to determine the timing of ovulation in relation to the LH peak after synchronization using PRID or Ovsynch protocols, to assess the effects of the period of treatment on these parameters and to provide information concerning how to use the two main protocols for fixed-time artificial insemination in buffalo. Forty-eight lactating Italian Mediterranean buffalo cows were used. The buffaloes were treated in various periods as follows: February to March (n = 12 PRID, n = 12 Ovsynch), end of the breeding season, May to June (n = 12 PRID, n = 12 Ovsynch), beginning of low-breeding season according to Italian environmental conditions. To determine the LH, blood samples were taken at 4-hour intervals, starting 24 hours from PRID removal (PRID group) or 12 hours from (PGF2α) injection (Ovsynch group) up to 108 hours. The ovaries were monitored by transrectal ultrasonography to verify ovulation. The LH–ovulation interval was similar in both groups (30.10 ± 1.05 and 32.77 ± 1.15 hours, respectively, in PRID and Ovsynch group). In the PRID group, the timing of ovulation in relation to device removal was 76.83 ± 3.65 hours with a high level of variability among the animals. In the Ovsynch group, we observed a better synchronization of LH peaks and ovulations, and the timing of ovulation in relation to the last GnRH injection was 35.67 ± 1.15 hours. The percentage of animals reaching the LH peak and ovulation was lower (P ≤ 0.05) in May to June (respectively 75.0% and 54.1%) compared to February to March (respectively 95.8% and 83.3%), indicating a reduction of hypothalamus–pituitary responsiveness to the synchronization treatments in the daylight-lengthening period.

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860). Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations. Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23–98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum). In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required.

The sub-Mediterranean climate, characterized by inter-annual variability, is undergoing an increase in aridity. We assessed how inter-annual variation of precipitation affects pastoral systems of central Italy, to evaluate whether climate change may cause extensive sheep farming to become unsustainable. Since the assessment of pasture-rumen interaction is considered a key tool in evaluating the status of the system, we collected data on precipitation, soil water deficit, aboveground phytomass, forage chemical composition, sheep rumen keratinization and body condition. We found that drought stress negatively affected forage quantity and quality with a pattern of responses that are partially different from those reported for both Mediterranean and Temperate regions. The most negatively affected plant communities were those of productive habitats. In clipped plots, we also observed a decrease in the re-growth capacity after clipping, especially in plant communities of southerly slopes. Positive correlations were identified for body condition with phytomass and crude protein, and negative correlations for rumen degree of keratinization with crude fibre and Acid Detergent Fibre. We suggested that the body condition of sheep deteriorates because the reduced quantity and quality of pasture changes the sheep rumen, which in turn limits absorption of volatile fatty acid.

Gastrin (GAS) and insulin (INS) play a significant role in the regulation of feed intake, as well as in the homeorhetic adaptations to different physiological stages, nutrient supply and requirements. The aim of the study was to verify if plasma GAS and INS concentrations in adult does are affected by the physiological state, change in response to the meal and are correlated with feed intake and energy balance. Dry matter intake (DMI) and pre and post-prandial plasma concentrations of gastrin-17 and insulin were recorded in 21 female goats fed hay and different quantities of concentrate (maize grain) twice daily in different physiological states. DMI was lower during the week of oestrous activity, compared to the period of anoestrus – progressively decreasing during pregnancy and increasing during the post-partum period, without significant differences between early and late lactation. Plasma GAS concentrations were significantly affected by physiological status, being higher (P < 0.05) during lactation than during pregnancy. Overall plasma GAS concentrations increased (P < 0.01) from pre-feeding levels (169 ± 24 pg/ml), to 30 min after feed ingestion (185 ± 29 pg/ml). The increase occurred 10 and 20 min (P < 0.05) after feeding, regardless of the diet composition. Both pre- and post-prandial GAS concentrations were related to DMI (P < 0.001), but not to the energy intake. Plasma INS on the other hand was not significantly affected by the physiological status. The pre-feeding plasma INS concentration, across all physiological states, was 1.36 ± 0.32 ng/ml and increased (P < 0.01) to 1.45 ± 0.38 ng/ml 30 min after feeding. Pre-feeding INS, but not post-prandial INS, increased with increasing proportions of maize in the diet and energy intake (P < 0.05). Overall INS was negatively correlated (P < 0.05) to DMI. It was concluded that cephalic reflexes are efficient stimula for both GAS and INS release in adult goats fed twice daily. Plasma GAS concentrations were related to hay intake and total DMI, thus with the volume of ingesta and presumably the time spent eating. GAS concentrations were not affected by the type of feed, nor the energy intake or the flux into the abomasum. Pre-feeding plasma INS was mainly dependent on the energy intake, while the post prandial increase was not dependent on the size or quality of the meal, or on the energy intake. Dietary manipulation and feed ingestion can thus affect the circulatory levels of these two main endocrine factors.

The effects of different concentrate levels in the diet on plasma 3-3′-5-triiodothyronine (T3) and thyroxine (T4) concentrations in does of different physiological conditions were investigated. Blood samples were taken starting in autumn from 20 goats fed hay ad libitum, during the dry period (30 days and 3 days prior to the synchronized oestrus; D1 and D2, respectively), pregnancy (week 13, week 17 and week 21 of pregnancy; P1, P2 and P3, respectively) and lactation (5 weeks and 13 weeks following parturition; L1 and L2, respectively). The control group (C, n = 10) was fed either 0.2 (in periods D1, D2 and P1) or 0.4 kg maize grain(head per day) (in P2, P3, L1 and L2). The high energy diet group (H, n = 10) was fed either 0.4 (in D1, D2 and P1) or 0.7 kg maize grain(head day) (in P2, P3, L1 and L2). Plasma total T3 concentrations were affected only by the sampling period, being significantly higher in the P1 period (0.96 ± 0.05 ng/ml) than in the dry period (0.72 ± 0.04 ng/ml) and in P3 (0.70 ± 0.02 ng/ml). In the C goats the plasma T4 concentration recorded in the P1 (65.6 ± 2.6 ng/ml) and L1 (62.8 ± 5.2 ng/ml) periods was significantly higher than that in P3 (40.2 ± 3 ng/ml). The variation in plasma T4 concentration in the H does was not significantly different throughout the different physiological stages. The decrease in plasma T3 and T4 concentrations in the second half of pregnancy in the H goats was delayed by 1 month. In addition, during the last 2 months of pregnancy, the higher concentrate diet induced higher plasma T3 (0.92 ± 0.06 ng/ml) and T4 (67.2 ± 6.6 ng/ml) concentrations when compared to the C (0.76 ± 0.04 and 45.1 ± 3.2 ng/ml for T3 and T4, respectively) group. The effect of energy intake on plasma thyroid hormone secretion during late pregnancy could be explained by a negative energy balance that usually occurs during this period. During lactation the higher energy diet had no significant effect on milk yield nor on the plasma thyroid hormone concentrations. The concentrate level of the C diet seemed to be sufficient to meet the energy requirements for milk production in these goats, as indicated also by the lack of body weight and body condition score (BCS) changes during lactation. The results of the present study highlight the relationships between thyroid activity and energy balance in the goat species. Circulating thyroid hormones represent a relevant metabolic index of the animals’ nutritional state.

The aims of the present work were to evaluate if a seasonality of plasma testosterone concentrations exists in Mediterranean male goats and it may be affected by climatic conditions. Testosterone concentrations were determined throughout 1 year in blood samples collected weekly from 24 males of 4 Mediterranean goat breeds (Ionica, Garganica, Maltese and Red Syrian), fed a constant diet, maintained without interactions with does and reared in 3 farms with different climates, in Italy. Plasma testosterone concentrations were affected by season (P0.0001), being higher during summer (8.33+/-0.35 ng/mL) than during autumn (6.81+/-0.26 ng/mL), which were in turn higher than in winter (1.55+/-0.08 ng/mL) and spring (2.04+/-0.15 ng/mL). An effect of breed was recorded (P0.001), Ionica bucks having the highest plasma testosterone means, Red Syrian and Maltese the lowest. The onset of the reproductive season was similar among the breeds. Ionica bucks had a later offset than Red Syrian and Maltese and a longer reproductive season than Maltese bucks (P0.05). No effect of farm nor interaction breed x farm was recorded. Single sporadic peaks of plasma testosterone were recorded in many bucks, outside the main reproductive season, especially during spring. In conclusion the bucks of all the four breeds displayed a clear seasonality of plasma testosterone, with very low levels from January to May and high levels from July to November; hormone levels and pattern of seasonality were affected by breed and not by climate.

The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalis bubalis). After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG) were enriched by Vicia villosa agarose (VVA) affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8 kDa and from 57.8 to 80.9 kDa in the mid- and late- pregnancy placenta respectively. Aminoterminal microsequencing of proteins allowed the identification of three distinct wbPAG sequences wich have ben deposed in the SwissProt database: RGSXLTIHPLRNIRDFFYUG (Acc. n. P85048), RGSXLTILPLRNIID (P85049) and RGSXLTHLPLRNI (P85050). Their comparison to those previously identified revealed that two of them were new since they have not been described yet. Our results confirm the suitability of VVA chromatography in enrichment of multiple PAG molecules expressed in buffalo placenta. Productions of specific antisera can be very useful in immonoistochemical and immunocyitochemical studies of PAG expression in fetomaternal interfaces. Purified native PAG are also required for development on specific immoassays (RIA/ELISA) currently used for pregnancy diagnosis and physiological investigation in farm animal.

Buffalo cows were submitted to three superovulatory treatments. T1 (n = 7): PRID for 10 days (d0-d9) plus decreasing doses of 500 IU FSH/LH (12 h-intervals d7‑d10); T2 (n = 8): PRID for 11 d (d0-d10) plus 2000 IU PMSG at d7; T3 (n = 9): PRID for 11 d plus 2000 IU PMSG at d7 and decreasing doses of 175 IU FSH/LH (12 h-intervals d10‑ d11). Overall plasma inhibin‑A (In-A) concentrations correlated with large follicles (LF, diameter >6mm, R=0.83, P10 mm at d12- 13 (T1=5.0+/-1.4, T2=1.2+/-0.9, T3=8.3+/-2.3). In-A concentrations significantly rised at d11-13 of T1 and T3. In-A seems a good indicator of the follicular development during superovulation in buffalo cows, while E2 is not. Furthermore T3 was followed by better ovarian follicular responses.

In Italian buffalo cows the spontaneous cyclic ovarian activity is mainly high in autumn, while during spring and early summer it is very low. However many farmers separate males from females in the October-February period to obtain births in winter-spring. In order to verify if blood testosterone concentration in adult buffalo bulls is affected by season and by different management of the contact with females, 20 adult buffalo males, bred in central Italy were submitted to monthly blood sampling for 1 year, from September to August. The bulls were kept together with females all the time (group A; n=9) or were held separated from cows from October to February (group B; n=11). The mean (+/-S.E.M.) serum testosterone concentrations were higher in spring and summer than in autumn and winter in group B (2.07+/-0.1 ng/mL versus 0.99+/-0.08 ng/mL, P

Since oestrus behaviour in buffalo is frequently scarcely clear and the oestrus signs, even when showed, are not reliable, ovulation can not be predicted. Furthermore high individual variability of the oestrus signs – LH peak interval was observed both in spontaneous and hormonal induced oestrus (Moioli et al., 1998; Barile et al, 1998).

In sub-Mediterranean climate the grassland aboveground phytomass production peaks in late spring and drops in summer, when the decrease of the pasture feed value may lead to the worsening of the animal welfare. Our goal was to define the summer rainfall values leading to a decrease of semi-extensive farming system sustainability in sub-Mediterranean regions. Summer rainfall variations reflect in the aboveground phytomass production and on the sheep body state. Differences of body condition score (BCS) among years were significant in late summer, which is the mating period for sheep. In the driest year the BCS of end August drops down to 2.1, largely below the value considered sufficient to ensure the animal breeding/milking performances (2.5). Reduction of summer rainfall greater than 15–20% compared to the normal average value (thus less than expected by the scenario of climate change) might be detrimental for semi-extensive rearing sustainability in sub-Mediterranean climate.

The traditional utilization of donkeys (Equus asinus) as dairy animals has recently attracted substantial scientific interest with regard to human nutrition. Donkey milk is well tolerated by infants with cows’ milk allergy, useful in the treatment of human immune-related diseases, in the prevention of atherosclerosis, and in-vitro studies showed an anti-proliferative effect. Active 3-3′-5-triiodothyronine (T3) in colostrum and milk could play different physiological roles, systemic and paracrine, for both the mother and the suckling offspring. The aim was to evaluate whether thyroid hormones (TH) concentrations in milk and blood of lactating donkeys change with the advancing lactation and whether they can be affected by dietary supplementation with several trace elements, some of them directly involved with TH synthesis (I), metabolism (Se) and action (Zn). Sixteen lactating jennies were divided into two groups (CTL and TE). Mixed feed for TE was added with Zn, Fe, Cu, Mn, I, Se and Co. Every 2 weeks milk and blood samples were collected at 11·00. Total concentrations of T3 in milk (T3M) and T3 and T4 in plasma (T3P and T4P) were assayed using ELISA kits, validated for the donkey species. T3M was not correlated with TH concentrations in blood, did not change with the stage of lactation, and was significantly higher in TE (4·09±0·07 ng/ml, mean±se) than in CTL group (3·89±0·08 ng/ml). T4P (81·8±5·2 ng/ml) and T3P (15·2±1 ng/ml) significantly changed with time, but were not significantly affected by dietary treatment. T3P/T4P ratio was significantly lower in TE group. This study indicates that in donkey milk the concentration of T3, a human-like bioactive compound, can be affected by trace elements intake.

Donkey's milk is well tolerated by human infants with cow's milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (−20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at −20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.

The collection of biological samples through non-invasive techniques represents one way of monitoring in vivo physiological changes associated with reproductive activity. Such techniques are particularly important for the study of animal species in the wild. The goals of this study were 1) to evaluate fecal progestogen (P), estrogen (E), and androgen (A) by means of radioimmunoassays, in male and female wild boars culled in the Piedmont, Italy area; 2) to compare them with plasmatic concentrations and the animals' reproductive status; and 3) to assess variations in reproductive seasonality between two populations of wild boars living in a mountainous vs. a plain habitat in Piedmont. The results demonstrate a positive correlation between fecal and plasmatic steroid concentrations (r=0.46, 0.58, and 0.45 for plasma P(4) and P, E(2) and E, and T and A; P0.05). Moreover, high fecal levels of both P and E (170 ng/g and100 pg/g respectively) were found in 70.6% of pregnant sows and in none of the non-pregnant animals, thus supporting the use of this technique for detecting pregnancy status in wild boar. Similar birth patterns were displayed by the mountain and plain populations, but births peaked significantly only in the mountain population, in the spring (46%, P0.05, vs. other seasons). A corresponding autumnal peak of plasma testosterone concentrations in males was displayed only by the mountain population (7.4 vs.2.0 ng/mL in the other seasons, P0.05). The correlation between fecal and plasmatic steroid concentrations obtained in this study supports the applicability of this non-invasive sampling technique for monitoring reproductive status in wild boar, thus enabling a more informed and correct management of the species.

The plasma melatonin nychtemeral profiles in Mediterranean ewes and goats were evaluated six times throughout the year. Melatonin levels were high throughout the night and generally below the assay detection limit during daytime. However, during long days, 30% of the last daytime samples had high melatonin concentrations. Plasma melatonin levels were higher in Comisana sheep than in goats, and higher in Maltese than in Red Syrian goats, with highly significant effect of the individual animal and high repeatability. Plasma melatonin was higher in April than in August. When there was a large difference between the duration of day and night, the plasma melatonin pattern and the light/dark cycle did not always match exactly, suggesting some form of superimposition and/or the prevalence of an endogenous rhythm. The difference found at similar scotoperiods with increasing or decreasing day length may be involved in the perception of the photoperiodic changes.

Summary Twenty-two Angora kids were used to study the effect of a dietary supplementation with field bean (Vicia faba L. var. minor) on hair follicle activity, mohair characteristics and plasma total thyroxine (T4), triiodothyronine (T3), insulin (INS) and insulin-like growth factor-1 (IGF-1) concentrations. At birth, their mothers were divided in two groups (S: supplemented, C: control), the S group being supplemented with 300 g/head/day of whole field bean. At weaning, diet of kids from S group was supplemented with 80 g/head/day of whole field bean. At weaning, secondary hair follicle activity (S: 0.91 ± 0.01, C: 0.84 ± 0.02, p

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

The aim was to evaluate the suitability of two commercial ELISA kits for human serum or plasma, to measure Inhibin-A (In-A) and Estradiol-17-beta (E2) concentrations in buffalo plasma. Blood samples were obtained by jugular venipuncture from buffalo heifers and cows, and plasma samples were stored at – 20°C until assays. Precision of the methods was evaluated by the intra- and inter-assay coefficients of variation (CVs) of buffalo plasma sample replicates, at different concentrations. Accuracy was evaluated calculating the recovery rates of different proportions of the highest standard added to a buffalo plasma sample at low concentration (observed/expected values x 100). Linearity was evaluated by serially diluting one buffalo plasma sample at high concentration with the assay buffer and calculating by regression analysis the parallelism of the resulting line with the standard line. Intra-assay CVs were 11% and 15.1% for In-A and 1.8% and 3.3% for E2. Inter-assay CVs were 13.9% and 7.4% for In-A and E2, respectively. Mean recovery rate was 97.9% and 98.5% for In-A and E2, respectively. Dilution tests gave good parallelism between the lines obtained and the standard lines. It is concluded that the kits tested are suitable and reliable for buffalo plasma samples.

Thyroid hormones (TH) are the primary endocrine stimulators of non-shivering thermogenesis and are known to stimulate lactation. Triiodothyronine (T3) is the bioactive form, mainly derived by deiodination of thyroxine (T4), and the free quote (unbound to plasma proteins) is immediately bioavailable. This study aimed to evaluate potential relationships among TH in the blood, triiodothyronine in the milk (T3M), milk yield and environmental temperature in March to July for 8 lactating donkeys. Milk yield and blood TH concentrations changed significantly over time, whereas T3M was rather stable among individuals and not affected by time of sampling. Free T3 was not correlated with free T4 or with total TH in the blood, but it was weakly correlated with T3M. No relationship was found between blood TH and milk yield, which was negatively correlated with T3M. Thus, the absolute quantity of bioactive hormone in milk secretion is maintained. Milk yield was positively correlated with the free/total T3 and free T3/free T4 ratios, thus in turn with the relative quote of the circulating bioactive hormone. Circulating T3/T4 ratios were negatively correlated with environmental temperature. It is concluded that environmental temperature, in the range of the present study (-2 to 35°C), does not significantly entrain thyroid gland activity, which is affected more by other factors, such as inter-individual variations and physiological status (i.e., stage of lactation). However, increases in environmental temperature most likely induce decreases in deiodinase activity at the peripheral tissue level, as indicated by the decrease in the T3/T4 ratios in the blood.

The aim of this trial was to evaluate the effectiveness (fertility and lambing) of priming with a single injection of progesterone plus PMSG in anovulatory lactating Sarda ewes subjected to the ram effect (RE) in spring. Thirty ewes (P4 group) were i.m. injected with 30 mg progesterone and 500 IU PMSG 36 h before ram introduction (d 0). This treatment was compared to a 12-day treatment with fluorogestone acetate intravaginal sponges that was followed by injections of 350 IU PMSG upon sponge withdrawal (FGA group, n=30). All ewes responded to RE, showing plasma progestrone concentrations1 ng/mL between d 6 and 12 (FGA) or 6 and 9 (P4). Eighty-nine percent of the P4 ewes conceived at first ovulation, and 11% conceived following a short estrus cycle. Lambings occurred on d 150.4 +/- 3.9, and the lambing rate was 100%. The fertility of the FGA ewes was 83% for the induced ovulation and was 7% for the second ovulation after a normal cycle. The FGA ewes lambed on d 149.8 +/- 4.4, and the lambing rate was 83%. Two abortions were recorded for the FGA ewes, which had higher prolificacy than the P4 group (2.2 +/- 0.8 vs. 1.8 +/- 0.4, respectively; P0.05). Both fertility and the lambing rate were high in both groups, with a high degree of estrus synchronization, and there were no significant differences between the groups. We concluded that priming of lactating Sarda ewes in spring with P4+PMSG before RE is an effective and competitive method (cheaper and more practical than FGA+PMSG) of inducing fertile ovulations in these ewes.

Thirty-two post-partum Mediterranean river buffalo cows were continuously observed for signs of oestrus from September to December with the aid of two vasectomized bulls. Symptoms of oestrus among female Mediterranean buffaloes are weak, therefore oestrus was assessed based on bull behaviour (following and licking a cow and trying to mount her). Oestrus was divided into three phases based on the bull behaviour assessment. Status of the uterus and ovarian follicles were checked rectally every day for each cow which was detected by a teaser bull to be in oestrus. All cows in oestrus were bred twice by artificial insemination (AI), the first at ovulation and the second (using a different bull) 22 h later.The average duration of interest shown by the bull towards a cow (from the very first to the last sign of interest) was 68 h and the average length of the three phases was: phase 1 = 21 (s.d. 29) h, phase II = 32 (s.d. 24) h and phase III = 15 (s.d. 15) h. Fifteen buffaloes were bled during oestrus, and LH profiles were determined. No differences were evident among oestruses followed by pregnancy (no. = 18) and the others (no. = 26) for the variables describing behavioural events. Neither intensity of the bull courtship, presence or clarity of mucous discharge, or housing system affected the success of AI. The only differences between pregnant and non-pregnant cows were in the timing between the LH peak and the end of phase II (2·4 v. 14·7 h, P < 0·001), end of phase III (22 v. 40 h, P > 0·05) and ovulation (25 v. 46 h, P < 0·05). Successful pregnancies occurred 34 (s.d. 14) h after the end of phase II. The endocrinology and behavioural patterns of buffalo reproduction need further research to clarify the reasons for non-optimal pregnancy rates after AI.