Short communication: Heat treatment and souring do not affect milk estrone and 17ß-estradiol concentrations.

The aim of our study was to establish whether heat treatment and souring of milk affect its estrone (E1) and 17ß-estradiol (E2) concentrations. Milk samples were collected from 10 Holstein cows in late pregnancy. Concentrations of E1 and E2 were measured in milk samples that were previously heated to 70 and 95°C for 5 min. Additionally, E1 and E2 concentrations were determined in the same milk samples after 2 d of spontaneous souring at room temperature, and these samples were compared with E1 and E2 levels in raw, unprocessed milk. Concentrations of both hormones were determined by commercial ELISA kits. Concentrations of E1 in unprocessed and processed milk (milk heated to 70 and 95°C and soured milk) were (mean ± SE) 47.25 ± 4.16, 44.84 ± 3.47, 41.00 ± 4.55, and 44.92 ± 3.91 pg/mL, respectively. Concentrations of E2 in the same milk samples were 36.11 ± 10.01, 32.46 ± 9.88, 31.78 ± 9.56, and 31.43 ± 8.00 pg/mL, respectively. Concentrations of E1 and E2 in heat-treated milk did not differ significantly from those in unprocessed milk. Similarly, E1 and E2 concentrations in soured milk did not differ significantly from those in unprocessed milk samples. These results indicate that E1 and E2 are stable in milk and that milk processing (heating and souring) does not influence their degradation. Therefore, E1 and E2 concentrations are expected to be similar between commercial full-fat milk and the raw milk from which it was produced.

Does closantel in therapeutic doses display thyroid hormone-like activity in sheep?

The aim of this study was to define the thyroid hormone-like activity of closantel in sheep by measuring some blood parameters that are known to be influenced by thyroid hormones triiodothyronine (T3) and thyroxine (T4). Our hypothesis was that, if closantel possesses thyroid hormone-like activity, its use under in vivo conditions will result in changes similar to those in hyperthyroidism. The study was conducted in 20 Jezersko-Solchava breed sheep. Blood sampling was performed before and 10 days after routine anthelmintic treatment with closantel. Complete blood count, plasma cholesterol, triglycerides, protein, and albumin levels, as well as those of serum T3 and T4, were compared before and 10 days after closantel administration. This routine anthelmintic treatment of sheep with closantel did not significantly influence hematological parameters, thyroid hormone levels, or most of the biochemical parameters. No evidence was found for thyroid hormone-like activity of closantel in sheep. However, significantly (P < 0.01) elevated levels of plasma triglycerides were present 10 days after closantel administration.

Long-term leptin fluctuations in female donkeys.

REASONS FOR PERFORMING STUDY: The interest in donkeys is growing due to their integration in the systems of ecological farming, among other reasons. OBJECTIVES: Due to limited reports on leptin concentrations in donkeys, the aim of the present study was to examine age-dependent and seasonal changes in the circulating leptin concentration in female donkeys (jennies) and thus contribute to knowledge about the physiological characteristics of this species. STUDY DESIGN: Prospective longitudinal study. METHODS: The study was performed over a year (September 2008 to September 2009) on 20 yearling and young adult (pregnant, lactating or barren) jennies aged 1-5 years at the onset of the study; the animals were kept on pasture from May to September and stabled for the rest of the year. Blood samples were taken monthly and analysed for serum leptin concentrations by a commercial radioimmunoassay kit. RESULTS: Circulating leptin concentrations in studied jennies were lower than those reported for donkeys and horses. Despite the tendency for lower values in yearling vs. young adult jennies, the age range of the examined animals was insufficient to confirm any age-related leptin variations. Significant seasonal leptin fluctuations with peak levels in late spring and the lowest levels in autumn months, correlated with photoperiod, were detected in yearling, barren as well as pregnant jennies. Therefore, it was impossible to identify any effects of gestation or lactation on leptin concentrations of jennies. CONCLUSIONS: The results of this study cannot be used as evidence of a causal relationship between the photoperiod and seasonal circulating leptin fluctuations in donkeys, but could reflect changes induced by various external or internal factors enabling adaptations of grazing animals in variable submediterranean environments.

Serum cortisol and haematological, biochemical and antioxidant enzyme variables in horse blood sampled in a slaughterhouse lairage, immediately before stunning and during exsanguination.

The aim of the study was to determine changes of serum cortisol and biochemical, haematological and antioxidant enzyme variables in the blood of horses sampled during the pre-slaughter period (in the lairage and in the stunning box) and during exsanguination. A total of 24 Slovenian warm-blooded horses were observed. Blood samples for determination of serum cortisol and biochemical, haematological (red blood cell count, haematocrit, haemaglobin concentration) and antioxidant enzyme (whole blood superoxide dismutase and glutathione peroxidase) variables were collected by venipuncture of the left jugular vein in the lairage pen, 60 min before stunning (lairage) and immediately before stunning (stunning box). At exsanguination, blood samples were collected from the wound at the time of jugular vein sticking. During blood collection in the lairage pen and in the stunning box, horses were gently restrained with a halter. They were stunned using a penetrating captive bolt pistol impelled by air and were bled by jugular vein sticking. Horses were physically active in the lairage pen and in the race before entering the stunning box. After stunning, the horses showed paddling movements with their legs. In horses, the plasma lactate and glucose concentrations, the serum potassium concentration, the activities of the serum muscle enzymes aspartate aminotransferase and creatine kinase, and values of most of the other biochemical (Table 1) and haematological variables (Table 2), were significantly (P < 0.05) higher at exsanguination, than in blood sampled while they were in the lairage and in the stunning box. The serum concentrations of cortisol and chloride and the activities of alanine aminotransferase and antioxidant enzymes were not significantly different between the pre-slaughter period and exsanguination. All selected blood variables were not significantly different between the lairge and the stunning box sampling time, indicating no physiological stress responses of the investigated horses to stressors, such as novelty of the pre-slaughter environment and handling, present in the slaughterhouse between the lairage and the stunning box. However, the significantly higher values, at exsanguination, for the plasma lactate and glucose concentrations, serum muscle enzyme activities and haematological variables, than during the pre-slaughter period, might partially be attributed to stimulation of the sympathetic nervous system, caused by stunning and bleeding.

Serum leptin concentrations in Lipizzan fillies.

To determine seasonal changes in circulating leptin concentrations, as well as leptin fluctuations in various patterns of seasonal ovarian activity, blood samples were taken in 107 Lipizzan fillies aged 1-4 years over the whole year from January to December. Serum concentrations were measured in samples obtained once a month (leptin) or twice a month (progesterone), and evaluated with respect to the age of animals (yearlings, 2-, 3- and 4-year olds) and the pattern of ovarian activity (anoestrous, seasonal cycle, prolonged cyclicity, continuous cyclicity). The mean and monthly leptin concentrations in 3- and 4-year olds were significantly higher than in yearlings and 2-year olds (p < 0.001). Circulating leptin levels in 3- and 4-year olds increased significantly in summer, to peak in August and then decline significantly (p < 0.001) in autumn. The fluctuations in younger fillies were less evident. The highest mean serum leptin concentrations were observed in fillies exhibiting continuous cyclicity and the lowest in those, exhibiting seasonal cyclicity (p < 0.001). In anoestrous fillies, leptin concentrations were lower than in fillies with prolonged or continuous cyclicity but higher than in fillies with seasonal cyclic activity (p > 0.05).

Onset of puberty and duration of seasonal cyclicity in Lipizzan fillies.

REASONS FOR PERFORMING STUDY: The onset of puberty and duration of seasonal cyclicity in Lipizzan fillies and young mares have been determined mostly by empirical data, but very little is known about the hormonal changes at puberty and during the seasonal cyclicity in this breed. OBJECTIVES: To determine the onset of puberty and the course of the cyclicity in young Lipizzan mares from age 1-4 years. METHODS: Blood samples were collected twice a month for 2 years from 46 Lipizzan fillies age 1-4 years. Progesterone concentrations, indicating ovarian activity, were measured using an EIA kit. Values above 6.4 nmol/l were taken to indicate luteal phase and those below 3.4 nmol/l, oestrus. RESULTS: Fifty percent of Lipizzan fillies entered puberty during the first spring after their birth at mean +/- s.e. age 15.25 +/- 0.31 months and 50% during the second spring, at age 25.2 +/- 0.57 months. For several fillies of all ages, manifesting anoestrus during the winter months, the oestrus started in March and reached a peak in June to September. The duration of the oestrus activity was age-dependent, at 2.37 +/- 0.62 months in yearlings and 8.56 +/- 0.46 months in fillies age 4 years. In December, >50% of fillies age 2-4 years still exhibited oestrus activity and some entered anoestrus in January the following year. A small proportion of fillies age 3-4 years remained cyclic during the whole year, indicating that factors other than photoperiod can be involved in regulating seasonal cyclicity. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results demonstrate the influence of seasonal factors on the onset of puberty and on the course of oestrus activity in the majority of Lipizzan fillies. However, certain exceptions were also demonstrated but reasons for the prolonged periods of oestrus activity in some Lipizzan fillies were not identified, and the role of the hormone leptin should be studied in future investigations.

Establishment of a pregnancy following intravaginal insemination with epididymal semen from a dog castrated due to benign prostatic hyperplasia.

Benign prostatic hyperplasia was diagnosed in an American Staffordshire Terrier of high breeding value presenting concurrent haematuria. Castration as a treatment was synchronized with the oestrus cycle of a bitch selected for insemination. After castration the cauda epididymis was flushed with Gent semen extender and collected spermatozoa were filtered and analysed by Hamilton Thorn computer assisted sperm analysis. A total of 7 ml semen containing 742 x 10(6) spermatozoa with 76.5% mean motility was used for insemination. Intravaginal insemination of the bitch was performed with an insemination catheter for dogs (Kruuse, Marslev, Denmark) on the day when plasma progesterone levels reached 9.9 ng/ml. Normal pregnancy without complications resulted in eight live-born puppies 63 days after insemination. This is the first report of a normal pregnancy and birth of puppies from a bitch inseminated with epididymal semen obtained from a dog affected by benign prostate hyperplasia.

The variations of white blood cell count in Lipizzan horses.

Total and differential leucocyte counts (lymphocytes, neutrophils, monocytes, eosinophils and basophils) were measured in 140 stallions, 101 mares and 25 foals of Lipizzan breed. The values fell in the normal ranges for warm-blooded horses. Differences between mares and stallions were not significant with the exception of foals, having higher white blood cell, neutrophil, lymphocyte, monocyte and basophil values in females than in males. Foals exhibited an age-related increase of total leucocyte count during the first 4 months of life, accompanied by a decrease in neutrophil and increase in lymphocyte and eosinophil counts. In mares and stallions, the total number of leucocytes, lymphocytes, monocytes and basophils significantly decreased but the number of neutrophils and eosinophils remained almost unchanged with age gain.

Postnatal hypothyroidism does not affect prepubertal testis development in boars.

Young boars were treated with propiothiouracil to induce hypothyroidism to examine its effects on postnatal testicular development. Treatments with 0.1% 4-propyl-2-thiouracil (PTU) in drinking water started after weaning, at 3 weeks of age and all boars were severely hypothyroid at 6 weeks of age as determined by measuring T3 and T4 in blood plasma. Boars were castrated at different ages up to 20 weeks and their testes used for histological and immunohistochemical analyses. Although small but significant reduction in testis weight was observed from 8 to 12 weeks of age, this was not accompanied by significant difference in testicular volume. By 20 weeks of age, at the beginning of puberty, the differences in testis weights between control and treated groups of boars disappeared suggesting there is no lasting effect of hypothyroidism on postnatal development of boar testis. Immunohistochemical staining was used to determine the presence of molecular markers in both Sertoli and Leydig cells. Again, there were no differences between testes from control and treated boars in the pattern or intensity of immunostaining using antibodies against 3beta-hydroxysteroid dehydrogenase, antimullerian hormone or proliferating cell nuclear antigen (PCNA). Immunostaining with antibodies against PCNA showed interesting results as it was observed that Sertoli cells still express this marker of proliferating cells at 14 weeks of age, later than previously suggested cessation of Sertoli cell proliferation. This study suggests that hypothyroidism in boars does not have similar effects on postnatal testis development as reported in some other species.

The influence of age and gender on haematological parameters in Lipizzan horses.

Haematological parameters [red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), haemoglobin concentration, mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC)] in resting Lipizzan horses were determined for 143 stallions, 104 mares and 25 foals. The mean RBC and WBC values in Lipizzans were in the lower part of the normal range for warm-blooded horses. The mean PCV, MCV and MCH values were higher, but the mean haemoglobin concentration and MCHC values were lower than reported for other warm-blooded horses. In foals, the mean RBC, WBC, PCV, haemoglobin concentration and MCHC values were higher, whereas MCV and MCH were lower than in older animals. Results indicating a significant decrease in WBC (P < 0.01) and an increase in MCV, MCH and MCHC (P < 0.05) with increasing age are consistent with some other reports on warm-blooded horses. The age-related variations in RBC and PCV were less marked. Contrary to some reports, RBC (P < 0.01), WBC and haemoglobin concentration (P < 0.001) were higher in Lipizzan stallions than in mares, but differences in MCHC, MCH and MCV were insignificant. The specific haematological values determined in Lipizzans are presumably a result of selection and should be taken into consideration when dealing with this race of horses.

Serum T3 and T4 concentrations in the adult rats treated with herbicide 2,4-dichlorophenoxyacetic acid.

The influence of sublethal doses of 2,4-dichlorophenoxyacetic acid (2,4-D) on serum T3 and T4 concentrations in Hsd Cpb: Wistar rats of both sexes was studied. The trial was performed on 24 males and females respectively, each divided into three groups of 8 animals (control, groups 1 and 2). Aqueous solution of the compound (11 mg/kg body weight--group 1 and 110 mg/kg body weight--group 2) or clean tap water (control group) was used. Aliquots of 2.4 ml/kg body weight were administered with a stomach tube from the 1st to 10th day of the experiment. Three days before the first treatment and on the 6th and 13th day of the experiment the serum T3 and T4 concentrations were determined by commercial radioimmunoassay kits (Byk-Sangtec Diagnostica), validated for rats. A significant decrease of serum T4 (P < 0.01) and T3 (P < 0.001) was determined in males of groups 1 and 2 during the experiment. On the 6th day of experiment serum T4 and T3 values were significantly lower (P < 0.001 and 0.01 respectively) in group 2 than in the controls and group 1 of both males and females. During the whole experiment serum T4 levels were lower in females than in males (P < 0.05).