Luca Todini

Thyroid hormones in small ruminants: effects of endogenous, environmental and nutritional factors

Appropriate thyroid gland function and thyroid hormone activity are considered crucial to sustain the productive performance in domestic animals (growth, milk or hair fibre production). Changes of blood thyroid hormone concentrations are an indirect measure of the changes in thyroid gland activity and circulating thyroid hormones can be considered as indicators of the metabolic and nutritional status of the animals. Thyroid hormones play a pivotal role in the mechanisms permitting the animals to live and breed in the surrounding environment. Variations in hormone bioactivity allow the animals to adapt their metabolic balance to different environmental conditions, changes in nutrient requirements and availability, and to homeorhetic changes during different physiological stages. This is particularly important in the free-ranging and grazing animals, such as traditionally reared small ruminants, whose main physiological functions (feed intake, reproduction, hair growth) are markedly seasonal. Many investigations dealt with the involvement of thyroid hormones in the expression of endogenous seasonal rhythms, such as reproduction and hair growth cycles in fibre-producing (wool, mohair, cashmere) sheep and goats. Important knowledge about the pattern of thyroid hormone metabolism and their role in ontogenetic development has been obtained from studies in the ovine foetus and in the newborn. Many endogenous (breed, age, gender, physiological state) and environmental factors (climate, season, with a primary role of nutrition) are able to affect thyroid activity and hormone concentrations in blood, acting at the level of hypothalamus, pituitary and/or thyroid gland, as well as on peripheral monodeiodination. Knowledge on such topics mirror physiological changes and possibly allows the monitoring and manipulation of thyroid physiology, in order to improve animal health, welfare and production.

Plasma melatonin in domestic female Mediterranean sheep (Comisana breed) and goats (Maltese and Red Syrian)

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.

Measurement of thyroid hormones in donkey (Equus asinus) blood and milk: validation of ELISA kits and evaluation of sample collection, handling and storage

Donkeys milk is well tolerated by human infants with cows 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.

Essential trace elements in milk and blood serum of lactating donkeys as affected by lactation stage and dietary supplementation with trace elements

The aim of this trial was to study the concentration of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), selenium (Se), cobalt (Co) and iodine (I) in milk and blood serum of lactating donkeys, taking into account the effects of lactation stage and dietary supplementation with trace elements. During a 3-month period, 16 clinically healthy lactating donkeys (Martina-Franca-derived population), randomly divided into two homogeneous groups (control (CTL) and trace elements (TE)), were used to provide milk and blood samples at 2-week intervals. Donkeys in both groups had continuous access to meadow hay and were fed 2.5 kg of mixed feed daily, divided into two meals. The mixed feed for the TE group had the same ingredients as the CTL, but was supplemented with a commercial premix providing 163 mg Zn, 185 mg Fe, 36 mg Cu, 216 mg Mn, 0.67 mg Se, 2.78 mg Co and 3.20 mg I/kg mixed feed. The concentrations of Zn, Fe, Cu, Mn, Se, Co and I were measured in feeds, milk and blood serum by inductively coupled plasma-MS. Data were processed by ANOVA for repeated measures. The milk concentrations of all the investigated elements were not significantly affected by the dietary supplementation with TE. Serum concentrations of Zn, Fe, Cu Mn and Se were not affected by dietary treatment, but TE-supplemented donkeys showed significantly higher concentrations of serum Co (1.34 v. 0.69 μg/l) and I (24.42 v. 21.43 μg/l) than unsupplemented donkeys. The effect of lactation stage was significant for all the investigated elements in milk and blood serum, except for serum manganese. A clear negative trend during lactation was observed for milk Cu and Se concentrations (-38%), whereas that of Mn tended to increase. The serum Cu concentration was generally constant and that of Co tended to increase. If compared with data reported in the literature for human milk, donkey milk showed similarities for Zn, Mn, Co and I. Furthermore, this study indicated that, in the current experimental conditions, the mineral profile of donkey milk was not dependent on dietary TE supply.

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes.

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.

LH peak and ovulation after two different estrus synchronization treatments in buffalo cows in the daylight-lengthening period

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.

Thyroid Hormones in Donkey Blood and Milk: Correlations with Milk Yield and Environmental Temperatures

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.

Validation of ELISA kits for determination of Inhibin-A and Estradiol-17-beta concentrations in Buffalo plasma

Abstract 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.

Minor and potentially toxic trace elements in milk and blood serum of dairy donkeys

The aim of this trial was to study the concentration of Ti, V, As, Rb, Sr, Mo, Cd, Cs, and Pb in donkey milk and blood serum. One hundred twelve individual milk and blood serum samples were collected from 16 lactating donkeys (Martina-Franca-derived population; 6 to 12 yr old; 3 to 7 parities; average live weight 205.4kg; 32 to 58 d after foaling at the beginning of the trial) during a 3-mo-long experiment. The samples were analyzed for the aforementioned elements by inductively coupled plasma-mass spectrometry. Feedstuff and drinking water were also analyzed for the investigated elements. Data were processed by ANOVA for repeated measures. Average milk concentrations (±SD) of Ti, Rb, Sr, Mo, Cs, and Pb were 77.3 (±7.7), 339.1 (±82.1), 881.7 (±270.4), 4.5 (±1.6), 0.49 (±0.09), and 3.2 (±2.7) μg/L, respectively. More than 80% of samples were below the limit of detection for V, As, and Cd in milk and for Cd, and Pb in blood serum. The lower bound calculated for milk V, As, and Cd was 0.03μg/L for the 3 elements, the upper bound was calculated at 0.23, 0.10, and 0.31μg/L and the maximum value was observed at 0.54, 0.15, and 0.51μg/L, respectively. The average milk concentrations of Ti, Rb, Sr, Mo, and Cs were 600, 458, 346, 16, and 294%, respectively, than those of blood serum. Yet, Cs concentrations were in the same order of magnitude in milk and serum. Moderate to strong positive and significant correlation coefficients were observed between milk and blood serum concentrations for Ti, Rb, Sr, and Cs. The effect of the stage of lactation was significant for all the investigated elements in milk and blood serum, but most of the elements showed only small changes or inconsistent trends, and only the concentrations of Rb and Sr showed decreasing trends both in milk and blood serum. The relationship between milk and blood serum element concentrations indicates that the mammary gland plays a role in determining the milk concentrations of Mo, Ti, Rb, Sr, Mo, and Cs. In the current experimental conditions, in agreement with the low levels in drinking water and feedstuff, donkey milk concentration of potentially toxic elements was very low and did not raise health concerns for human consumption.

Follicular development, plasma Inhibin-A and Estradiol-17-beta concentrations in Buffalo cows during different treatment schedules for MOET programs

Abstract 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, P<0.01) and small follicles (SF, <6mm, R=-0.21, P<0.01). Plasma E2 correlated weakly with LF (R=0.21, P<0.05). Within treatment, there was always a positive correlation between In-A and LF (T1 R=0.83; T2 R=0.81; T3 R=0.83; P<0.01), but only in T3 there were correlations between In-A and E2 (R=0.67, P<0.01) and SF (R=-0.42, P<0.01), between E2 and LF (R=0.39, P<0.01) and SF (R=-0.35, P<0.05). T3 was followed by a higher number of follicles with diameter >10 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.