Dragan Gvozdić

ADRENOCORTICAL AND THYROID FUNCTION, HORMONE AND METABOLITE PROFILES AND THE ONSET OF OVARIAN CYCLICITY IN DAIRY COWS SUFFERING FROM VARIOUS FORMS OF KETOSIS

The involvement of adrenocortical and thyroid hormones in the pathogenesis of ketosis, as well as the ovarian consequences of this metabolic disorder, were studied in _2 parity cows (n=199) in 3 large scale dairy herds. To compare the plasma/serum concentrations of certain hormones Scortisol, thyroxin (T4), triiodothyronine (T3), insulin, insulin-like growth factor-1 (IGF-1)¹ and metabolites Sglucose (G), acetoacetic acid (ACAC), βOH-butyrate (BHB), non-esterified fatty acid (NEFA), trigliceride (TG), total cholesterol (TCh)¹, and the activity of aspartate aminotransferase (AST), blood samples were taken 1 to 3 days after calving and again 4 times 7 days apart. The ACTH-challenged cortisol responsiveness and the TRH-induced T4/T3 increase were determined between days 1 to 3 and again between days 28 to 35. The resumption of ovarian cyclicity was followed up by individual progesterone (P4) profiles based on milk samples taken 3 times a week for about 80 to 85 days. BHB level of 1 mmol/L was estimated as a border line between hyper- (>1 mmol/L) and normoketonaemic (<1 mmol/L) conditions. Five different ketone patterns were distinguished: (1) non-ketotic (n=98; normoketonaemia in all samples), (2) early type ketosis (n=45; hyperketonemia was detected only in the first week after calving), (3) late type (lactational) ketosis (n=11; after a normoketonaemic period increasing hyperketonaemia was detected in the 5th, or in the 4th and 5th weeks), (4) temporary ketosis (n=11; hyperketonaemia was detected for 1-2 weeks in the 2nd and 3rd or in the 3rd and 4th weeks); (5) long-lasting ketosis (n=34; hyperketonaemia has been detected since calving for 4 to 5 weeks or until dying / emergency slaughtering). Simultaneously with the hyperketonaemic stage increased NEFA, ACAC, depressed TCh, glucose and decreased insulin, IGF-1, T4 and T3 concentrations were detected in almost all the cases. Obvious metabolic and endocrine alterations were found, however, only in long-lasting ketosis. The TRH-stimulated T4 and T3 responses remained almost unaffected proving intact thyroid function in early and late type as well as in temporary ketosis. Depressed thyroid response and delayed onset of cyclic ovarian function were detected only in cases of long-lasting ketosis. The cows characterized by lower than normal (<mean-SD of non-ketotic cows) ACTH-stimulated cortisol response on days 1-3 after calving showed poorer chance for spontaneous recovery. There was a significant negative correlation between the IGF-1 level in the 1st week after calving and the duration of the postpartum acyclic period. In late type (lactational) ketosis the cessation of ovarian cyclicity was the most characteristic genital malfunction.

Ovarian activity and uterus organometry in delayed puberty gilts

About 30% of the total number of gilts selected for reproduction at the large breeding farm units in Vojvodina (Republic of Serbia) are culled due to prolonged pre-insemination anoestrus (estrus not detected until 8 mo of age). The aim of this study was to provide the answer to the following question: do the culling gilts reach cyclic ovarian activity at all? One hundred seventy five culled gilts in which external estrus manifestations were not detected by 8 mo of age were sacrificed and their reproductive organs were examined for determination of sexual maturity (ovaries exhibiting pre-ovulatory follicles 8 to 11 mm in diameter, corpora hemorrhagica, corpora lutea and corpora albicantia). Uterine weights and horn length were also determined. Functional ovaries were observed in 107 (61.1%) examined gilts, with 62 animals having one and 45 having two puberty ovarian cycles (57.9% and 42.1%, respectively). Pathomorphological changes which could result in prolonged pre-insemination anoestrus were not observed on the reproductive organs of sexually mature gilts. Our results indicate that most of the culling gilts have reached cyclic ovarian activity. The main reason for culling due to the absence of external estrus manifestations in sexually mature gilts could be inadequate estrus detection technology.

Effects of Different Amounts of Supplemental Selenium and Vitamin E on the Incidence of Retained Placenta, Selenium, Malondialdehyde, and Thyronines Status in Cows Treated with Prostaglandin F2α for the Induction of Parturition

The incidence of retained placenta (RP) in cows increases in cases of parturition induced by prostaglandin F2α. We analyzed the effects of different doses of supplemental selenium and vitamin E on the incidence of RP, blood selenium, plasma thyronines, and malondialdehyde concentration. Thirty-three clinically healthy, multiparous Holstein-Frisian cows were assigned to 3 groups and supplemented with a single intramuscular injection of sodium selenite (SS) and tocopherol acetate (TAc) between days 250 to 255 of gestation: control—unsupplemented; group A—10 mg SS + 400 mg TAc; group B—20 mg SS + 800 mg TAc. Parturition was induced using PGF2α not before day 275 of gestation. The RP incidence was reduced from 66.7% in the control to 38.2 and 30.8% in groups A and B, respectively. Blood selenium and glutathione peroxidase activity in treated groups were significantly higher compared to control, with no significant difference between groups A and B. Plasma malondialdehyde in group B was significantly lower than that in control and group A, while thyronines levels were not affected. Comparison of RP and non-RP cows, independently of supplement treatment, revealed higher blood selenium and glutathione peroxidase activity and lower MDA and thyroxine in non-RP animals, while triiodothyronine level did not differ.

Endocrine and metabolic profile in Holstein and red Holstein heifers during peripartal period

The aim of this stady was to examine the relationship between triiodothyronine (T3), thyroxin (T4), insulin-like growth factor-I (IGF-I) and blood serum liver function biomarkers concentrations in Holstein and Red Holstein heifers during the peripartal period. In the first experimental group (I) were heifers of Holstein Friesian breed (n=10), and in the second experimental group (II) were Red Holstein heifers (n=10). Blood samples were taken from the vena coccygea three times during the short peripartal period (5 days before delivery, at delivery and 5 days after delivery), and blood serum samples were separated after spontaneous coagulation. Blood serum T3, T4 and IGF-I concentrations were determinated using radioimmunoassay (RIA). Blood serum concentrations of glucose (G), total protein (TP), albumin, (Alb), urea, total bilirubin (tBil) and aspartate aminotrasferase (AST) were determined using commercial ELISA kits. The statistical significance between means was determined using Student t-test. Blood serum T3 concentration in Group II was significantly lower after delivery, and T4 concentration was significantly higher before delivery (p<0.05), compared to Group I. Mean blood serum IGF-I concentrations between the two experimental groups were not significantly different at all time intervals. Relationships between the determined hormones showed a median positive correlation between T3, and T4 (r=0.74) in Group I and highly positive correlation in Group II (r=0.81). Blood serum thyroid gland hormones and IGF-I concentrations in healthy heifers during peripartal period vary considerably, the lowest values being determined immediately after calving in both experimental groups. Blood serum G, TP, Alb and urea concentrations were significantly higher in Group I at all investigated time intervals. Blood serum tBil concentration was significantly higher after partus in Group I (11.02 ± 3.33 : 8.49 ± 3.35 μmol/L, Group I vs. II, respectively), and AST activity was not significantly different at all intervals.

The serum levels of insulin and igf-i in newborn piglets treated with clinoptilolite

The aim of this study was to investigate the influence of the natural mineral adsorber clinoptilolite on the serum levels of insulin and insulin-like growth factor-I in newborn piglets in the first 30 hours postnatally. A total number of 40 crossbred Landrace x Duroc newborn piglets from 4 litters was used. Five piglets from each litter were randomly assigned to the treated group and another five piglets to the control group. The treated animals received 10 ml of 15% clinoptilolite suspension three times: immediately after birth and subsequently at 12 and 24 hours after birth. Mean serum insulin level in the treated newborn piglets was almost 20% higher at both time intervals after treatment but the increases were not statistically significant, due to the high individual variation (44.40±6.33:36.40±5.14 and 17.54±2.61:14.02±1.14 mIU/L, treated vs. control at 10 and 30 hours postpartum). Serum levels of IGF-I were also increased in the treated newborn piglets, and the differences between means were statistically significant (18.20±0.63: 13.70±1.02 and 17.61±0.173:12.48±0.64 nmol/L, p<0.001, treated vs. control at 10 and 30 hours postpartum). Our results indicate that clinoptilolite treatment could effectively increase serum IGF-I and possibly also insulin levels in newborn piglets.

Supplemental Selenium Reduces the Levels of Biomarkers of Oxidative and General Stress in Peripartum Dairy Cows / Dodati Selen Snižava Nivoe Biomarkera Oksidativnog I Opšteg Stresa Kod Mlečnih Krava U Peripartalnom Periodu

The aim of this investigation was to determine the influence of oxidative stress upon general stress in dairy cows on parturition and whether the indicators of stress can be reduced by selenium (Se) supplementation. A total of 36 animals were divided into 3 groups 21 days prepartum and treated with a single-term intramuscular supplement of sodium selenite: Control group - 0 mg; group Se10 - 10 mg; group Se20 - 20 mg. Se supplementation significantly raised blood Se content and glutathione peroxidase (GPx) activity in groups Se10 and Se20, compared to Control, although there was no marked difference between supplemented groups. Plasma malondialdehyde (MDA) and cortisol concentrations were significantly reduced in supplemented groups Se10 and Se20, compared to Control. A negative correlation was detected between blood GPx activity and plasma MDA, while a positive correlation was determined between plasma MDA and cortisol concentrations. These results indicate that prepartum Se supplementation can be utilized for a partial relief of stress in cows during labor by augmenting the antioxidative action of GPx. Cilj ispitivanja je bio da se odredi obim uticaja oksidativnog stresa na opšti stres kod mlečnih krava u toku 24 sata oko porođaja, kao i da li on može biti ublažen primenom selena. Ukupno 36 krava je bilo podeljeno u tri grupe 21 dan pre porođaja i jednokratno tretirano natrijum selenitom aplikovanim intramuskularno u dozama: kontrolna grupa - 0 mg; grupa Se10 - 10 mg; grupa Se20 - 20mg. Grupe Se10 i Se20 su imale značajno višu koncentraciju selena i aktivnost glutation peroksidaze (GPx) u krvi u odnosu na kontrolnu grupu, iako nije uočena razlika između suplementiranih grupa. Koncentracije malondialdehida (MDA) i kortizola su bile značajno niže u grupama Se10 i Se20 u odnosu na kontrolnu grupu. Uočena je negativna korelacija između aktivnosti GPx u krvi i koncentracije MDA u plazmi, a pozitivna korelacija između koncentracije MDA i kortizola u plazmi. Rezultati ukazuju da prepartalna upotreba selena može biti korisna u smislu delimičnog smanjenja stresa u toku porođaja zbog povećanja antioksidativnog delovanja GPx.

Fertility of sows after intracervical or intrauterine insemination with different spermatozoa number in reduced volume doses.

Modern intensive pig production demands an increasing number of insemination doses per ejaculate of genetically superior boars. In order to achieve such a result the possibility of producing insemination doses in both reduced volume and spermatozoa count without decreasing the fertility of sows is studied. In this trial we studied the effect of insemination with reduced volumes of semen (50 mL) and varied spermatozoa count (4, 2 or 1x109). Insemination was performed by the classical (intracervical) or by the new (intrauterine) techniques and the basic fertility parameters (farrowing % and litter size) were measured. The farrowing value decreased with decreasing spermatozoa numbers after intracervical and intrauterine insemination. However, the farrowing value, regardless of spermatozoa numbers, was significantly higher after intrauterine insemination (83.3%, 76.7%, 66.7%) compared to the classical intracervical insemination (73.3%, 66.7% and 50%). Litters size did not vary significantly depending on the applied insemination procedure or spermatozoa number. These results indicate that application of the new intrauterine insemination procedure enables the use of doses with a smaller volume and spermatozoa number, at the same time obtaining a satisfactory farrowing and litter size. This opens the possibility of obtaining a significantly higher number of doses per ejaculate when compared to the classical intracervical insemination procedure. In such a way can be increased significantly the degree of reproductive exploitation of genetically superior boars.

Blood serum free amino acids pattern in newborn calves on colostral diet and orally treated with zeolite.

Oral zeolite treatement effects on the blood serum free amino acid pattern in newborn calves was investigated. The total number of 30 newborn Holstein calves of both sexes, weighting 35±3 kg (mean±SD), were immediately after parturition separated from their dams and placed in individual pens. Calves were divided in two experimental groups, 15 calves each. All calves were bottle-fed twice/ day (1.5 L/meal) during the first 48 hours after delivery, in 12 hour intervals, with their mothers first (during 24 hours postpartum) or second colostrum (at 24-48 hours postpartum), starting two hours after delivery. Zeolite suspension (20 mL, 25% suspension in distilled water) was added to every meal for treated calves. Colostrum samples were collected from six cows at 0-12h and 24h after delivery. Total and colostral whey protein concentrations were determined using the colorimetric method. Blood samples were taken from the jugular vein of calves at 6, 16, 30 and 40 hours after birth. After spontaneous coagulation at room temperature blood serum was separated and stored at -20°C until analyzed. Total protein concentration was determined by the colorimetric method. Blood serum immunoglobulin G (IgG) concentrations were determined using single radial immunodiffusion (sRID) plates. Pooled blood serum free amino acids (aspartic acid - Asp, glutamic acid - Glu, serine - Ser, histidine - His, glycine - Gly, threonine - Thr, alanine - Ala, proline - Pro, tyrosine - Tyr, arginine - Arg, valine - Val, methionine - Met, Leucine - Leu, Isoleucine - Ile, phenylalanine - Phe) were identified and quantified using high-performance liquid chromatography (HPLC, GBC Australia). Total and colostral whey protein concentrations were significantly higher in the first colostrum and decreased between 50-75% at 24-48 hours later on. Mean blood serum IgG concentration was significantly increased at 6 and 16 hours in the treated calves (26±7:20±5 and 55±15:42±13 g/L, p<0.05). Blood serum free amino acids (AA) first were separated at nonessential and essential AA (NEAA and EAA, respectively), both being increased at all time intervals after birth in treated calves. However, when the pooled blood serum free AA were clustered according to polarity and electrical charge and presented as relative values (% of the control group values) at the 6h there was a massive increase of polar positive (Arg, His), polar neutral (Ser, Thr, Tyr) and nonpolar neutral free AA (except Met). The minimal effect of oral zeolite treatment was on the negative polar blood serum free AA concentration (Asp and Glu).

Brix Refractometry of Colostrum from Primiparous Dairy Cows and New-Born Calf Blood Serum in the Evaluation of Failure of Passive Transfer

Abstract Failure of passive transfer (FPT) of immunoglobulins (IgG) is associated with increased morbidity and mortality of calves. In this study we evaluated the digital Brix refractometer usefulness for the assessment of FPT. A number of 16 colostrum samples from the first milking (2-3h post-partum) of Holstein-Friesian dairy heifers and 29 blood sera of 3-6 days old calves were analyzed with a digital Brix refractometer. Total proteins were determined by the biuret reaction. Colostral IgG were determined by radial immunodiffusion (RID), and colostral whey and blood serum γ globulin (composed almost entirely of IgG) were determined by agarose protein gel electrophoresis (APE) and densitometry. Colostral % Brix score was 25.5±3.4%. Concentrations of colostrum IgG and colostral whey γ globulin were 130±33 g/L and 100±24 g/L respectively. The concentration of total proteins in colostral whey was 134±30 g/L. The correlations between Brix values and the concentrations of IgG determined with RID and the concentrations of γ globulin determined with APE were positive and highly significant (P<0.001 and P<0.01). The concentration of serum proteins of new-born calves was 57.75±11.8 g/L, the concentration of γ globulin was 14.4±7.8 g/L, and the Brix score was 8.6±1.0%. FPT (serum γ globulin<10 g/L) was detected in 34.5% (10/29) calves. Brix score correlated with the concentration of blood serum γ globulins in all examined calves. The results have confirmed that digital Brix refractometry allows the producers to use this technique in order to estimate colostral and calf serum IgG, thereby monitoring both colostrum quality and success of passive transfer.

Effects of Selenium and Thyroid Hormone Deficiency on Peritoneal Macrophages Adhesion and Occurrence of Natural IGM Antibodies in Juvenile Rats

Abstract Both selenium, as an effector and regulator of antioxidative enzymes activity, and thyroid hormones are potent immunomodulators. Besides, selenium incorporated into iodothyronine deiodinases is involved in the thyroid function and thus indirectly regulates the immune response. Studies of the mutual influence of selenium and thyroid hormones on the immune response are scarce, hence we analyzed the effects of an iodothyronine deiodinases blocker, propylthiouracil (PTU), and selenium deficiency on the function of peritoneal macrophages, and titer of naturally occurring anti-sheep red blood cells (SRBC) IgM antibodies in juvenile rats. The experiment was carried out on 64 Wistar male rats allotted to 4 groups: controlselenium adequate PTU- group; selenium adequate, PTU+ group; selenium defi cient, PTU- group; and selenium defi cient, PTU+. The selenium adequate and selenium defi cient groups were fed a diet containing 0.334 and 0.031 mg Se/kg, respectively. PTU+ groups received PTU (150 mg/L) in drinking water. After 3 weeks, thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) were determined. The animals having “intermediate” concentrations of T3 (1.56-1.69 nmol/L) and T4 (41-50 nmol/L) were excluded from further analysis. Thus, PTU+ groups included hypothyroid animals (T3≤1.55 nmol/L; T4≤40 nmol/L), while PTU- groups included euthyroid rats (T3≥1.70 nmol/L; T4≥50 nmol/L). Both groups of selenium deficient rats had, when compared to the control group, a significantly lower activity of glutathione peroxidase GPx1 and GPx3. Neither selenium deficiency nor PTU influenced the adherence of peritoneal macrophages. Selenium deficiency significantly decreased the peroxide synthesis in macrophages and significantly increased the titer of anti-SRBC IgM. Hypotyroidism alone or in combination with selenium deficiency had no influence on these parameters.