Oxidative state in the estrus cycle of the buffaloes: a preliminary study.

The oxidative status, the relationship between oxidizing agents (free radicals) and antioxidant system, is able to influence the correct performance of the estrus cycle. The aim of the experimentation was to define a first range of physiological values of reactive oxygen species (ROS) and the biological antioxidant potential (BAP), found during the phases of the buffalo estrus cycle. In this study, blood samples were taken from 30 buffaloes in various phases of the estrus cycle (estrus, diestrus, anestrus), on which serum determinations, by a photometer, of BAP and ROS have been carried out. The highest ROS values were detected during estrus and this is due to the primary role they play in determining ovulation. The highest levels of BAP have been found in diestrus, when they perform protective action against oxidative damage in the ovaries and uterus.

Oxidative stress in neonatology: a review.

Free radicals are highly reactive oxidizing agents containing one or more unpaired electrons. Both in human and veterinary neonathology, it is generally accepted that oxidative stress functions as an important catalysator of neonatal disease. Soon after birth, many sudden physiological and environmental conditions make the newborn vulnerable for the negative effects of oxidative stress, which potentially can impair neonatal vitality. As a clinician, it is important to have in depth knowledge about factors affecting maternal/neonatal oxidative status and the cascades of events that enrol when the neonate is subjected to oxidative stress. This report aims at providing clinicians with an up-to-date review about oxidative stress in neonates across animal species. It will be emphasized which handlings and treatments that are applied during neonatal care or resuscitation can actually impose oxidative stress upon the neonate. Views and opinions about maternal and/or neonatal antioxydative therapy will be shared.

Oxidative stress during pregnancy in the sheep.

During physiological pregnancy, all tissues and, mostly, placenta and foetus require high amounts of oxygen. Reactive oxygen species (ROS), generated both by mother and foetus, are implicated in foetal growth because they promote replication, differentiation and maturation of cells and organs. Nevertheless, ROS excess, if not properly counterbalanced, may lead to an alteration in cell constituents, with harmful effects both on mother and foetus.ROS exert a biphasic effect because adequate ROS concentration is essential for embryo development, implant, foetal defence against uterine infections, steroidogenesis, pregnancy maintainance and partum. On the other hand, an uncontrolled ROS generation, beyond physiological antioxidant defences, may lead to embryo resorption, placental degeneration with subsequent alteration in maternal-foetal exchanges, delay in foetal growth, pregnancy interruption, stillbirths. This review investigates the mechanisms underlying ROS generation and effects, throughout physiological and pathological pregnancy in sheep, with a look to antioxidants and their importance in such a critical phase of the reproductive cycle of the sheep.

Alpha-fetoprotein in animal reproduction.

Alpha-fetoprotein (AFP) is a serum glycoprotein with structural and physico-chemical properties similar to albumin. However, the exact physiological functions of AFP remain unknown; those known to date include markers to pathological conditions including neoplastic and non-neoplastic diseases, antioxidant effects, growth regulator in different cells and in cancer, immune response modulator, and carrier for fatty acids and oestrogens. This review aimed to present an overview of the different functions of AFP, particularly its role in the sexual differentiation of the hypothalamus, because its ability to bind oestrogens prevents their passage to the brain, where they inhibit the surge centre development. AFP and anti-Mullerian hormone are known to be involved in the development of freemartins, or genetically female foetuses masculinised in the presence of a male co-twin.

Intra-vesicle administration of D-cloprostenol for induction of abortion in mid-gestation bitches.

This study evaluated a new method for mid-gestation termination in the bitch, which consisted of ultrasound-guided administration of a single dose of d-cloprostenol, a PGF(2alpha) analogue, into a single gestational sac. Effects on serum progesterone concentration (P(4)) were also investigated. The study was performed between days 28 and 35 of gestation on 15 privately owned crossbred bitches, randomly divided into two groups: group A comprised 10 bitches treated with 15 microg per head d-cloprostenol diluted in 0.8 ml sterile saline (final volume 1 ml); group B comprised 5 bitches treated with 1 ml of sterile saline solution (0.9% NaCl), administered in the same way. In all bitches of group A, fetal death was successfully induced within 5 days (mean: 3.1 days, S.D. 1.2) with no clinical or behavioural complications. Mild adverse effects were observed in two bitches, each weighing less than 10 kg, including salivation, defecation and hyperventilation, which disappeared within 15 min. None of the subjects in group B aborted within 10 days post-treatment. In group A, P(4) declined 2.8 days before pregnancy termination to a mean value below 30 nmol/l (S.D. 2.9 nmol/l). However, two bitches showed a higher concentration of P(4) throughout the sampling period. Our study demonstrates that intra-vesicle administration of a single low dose of D-cloprostenol is an effective and safe technique for induction of abortion, which offers an additional option for termination of unwanted pregnancy in the mid-gestation bitch.

The effect of calcium-naloxone treatment on blood calcium, beta-endorphin, and acetylcholine in milk fever.

Milk fever is a postpartum syndrome of cows characterized by acute hypocalcemia, which reduces the release of acetylcholine (ACH), inducing flaccid paralysis and recumbency. Our aim was to evaluate the effect of calcium (Ca2+) combined with naloxone (Nx, an opioid antagonist; Ca2+-Nx) on plasma concentrations of ACH, beta-endorphin (betaE), and Ca2+ just before treatment (T0) and at 15, 30, and 90 min after treatment (T15, T30, and T90, respectively). Thirty cows were divided into 3 groups of 10 cows each. In group A1, cows affected by milk fever were treated (i.v.) with a combination of 0.2 mL/kg of body weight (BW) of Ca2+ borogluconate (20%) and 0.01 mg/kg of BW of Nx hydrochloride dihydrate. In group A2, cows affected by milk fever were treated (i.v.) with 2 mL/kg of BW of Ca2+ borogluconate (20%). In group C, healthy cows were treated (i.v.) with a combination of 0.2 mL/kg of BW of Ca2+ borogluconate (20%) and 0.01 mg/kg of BW of Nx hydrochloride dihydrate. Cows underwent treatments within 24 h of calving. Blood samples were collected at T0 and at T15, T30, and T90 for quantitative determination of ACH, betaE, and Ca2+. The cows in groups A1 and A2 recovered within a mean of 20 +/- 10 min, although 4 cows in group A2 underwent a relapse. Blood Ca2+ concentrations in group C increased slightly at T30 and at T90 (T30: 8.8 +/- 0.6 mg/dL; T90: 8.7 +/- 0.6 mg/dL) after treatment, whereas the response in groups affected by milk fever was similar, even though Ca2+ concentrations showed a sharp increase (A1: 8.9 +/- 0.8 mg/dL; A2: 6.0 +/- 0.7 mg/dL), particularly at T15 in group A1. Concentrations of betaE showed a similar pattern in groups A1 and C, with an increase at T15 (A1: 8.2 +/- 1.0 ng/mL; C: 2.7 +/- 0.4 ng/mL) and a subsequent decrease until T90 (A1: 1.4 +/- 0.3 ng/mL; C: 1.4 +/- 0.4 ng/mL), whereas betaE remained constant throughout in group A2. Concentrations of ACH in group A1 decreased significantly between T0 and T15, T30, and T90 (T0: 7.2 +/- 1.1 nmol/L; T15: 4.2 +/- 1.2 nmol/L; T30: 2.9 +/- 0.8 nmol/L; T90: 3.1 +/- 0.3 nmol/L), whereas in group A2, it did not change. In group C, concentrations of ACH decreased at T15 and increased again at T30 (T15: 1.1 +/- 0.3 nmol/L; T30: 3.2 +/- 0.7 nmol/L). Our results suggest that administration of Ca2+-Nx, which restored the physiological Ca2+ concentrations, might have an effect on nicotinic receptors by restoring the normal neuromuscular transmission at the motor endplate.

Lidocaine decreases the xylazine-evoked contractility in pregnant cows.

The objective of this in vitro study was to evaluate and compare the effects of xylazine on basal uterine contractility of bovine pregnant uterine strips and that of lidocaine on xylazine-sensitized bovine pregnant uterine strips, at different stages of pregnancy. Basal contractility was evaluated in an isolated organ bath and the functionality of the strips throughout the experiment was evaluated using a dose of carbachol (10(-5)M). Uterine motility, expressed with amplitude, frequency of contractions as well as the area under the curve, was recorded in different stages of pregnancy and data were collected at 15-min intervals (5-min before and 5-min after xylazine administration and 5-min after lidocaine addition on the plateau contraction induced by xylazine). Uterine motility increased in all the stages of pregnancy after xylazine addition and gradually decreased after treatment with lidocaine. These data suggest that lidocaine might decrease the tonic effect induced by xylazine on bovine pregnant uteri.

Cystic ovarian follicles and thyroid activity in the dairy cow.

Thyroid activity affects the functionality of the reproductive axis and thyroid dysfunction has been associated with ovarian hyperstimulation syndrome and polycystic ovarian syndrome, in human medicine. This study investigates serum17- estradiol, progesterone, thyrotropic and thyroid hormone levels, in cyclic dairy cows on heat (Group H) and in dairy cows with ovarian follicular cysts (Group FC). Both 17- estradiol and progesterone serum concentrations were statistically higher in cystic than in cyclic cows (estradiol: 8.51±1.91 vs 6.32±1pg/mL) (progesterone: 0.49±0.17 vs 0.13±0.03ng/mL), whereas TSH and fT4 serum concentrations were statistically lower in cows with cystic ovarian follicles (COF), compared to cyclic ones (TSH: 2.48±1.31 vs 3.56±1.03ng/mL) (fT4: 5.86±1.69 vs 8.63±1.08). fT3 serum levels were similar, in both cystic and cyclic subjects (2.94±0.65 vs 3.02±0.9, respectively). Based on these results it was decided to examine the function of the thyrothropic axis of dairy cows in a similar manner to that conducted on humans. If severe hypothyroidism should be found, a hormone replacement therapy could be attempted in cystic cows refractory to "ordinary" therapies.

Blood and milk oxidative status after administration of different antioxidants during early postpartum in dairy cows.

This paper describes serum and milk oxidative profiles during early postpartum (10th-16th day) of dairy cows, after three different treatments: ß-carotene and α-tocopherol (Dalmavital®, Fatro, Italy) (VAE Group); L-carnitine, d, L-acetylmethionine, cyanocobalamin (Adiuxan®, Ceva Vetem, Italy) (LCAR Group) and Dalmavital®+Adiuxan® (VAE/LCAR Group). Antioxidant administrations resulted in a general reduction in ROS concentrations which decreased more after a single drug administration (VAE and LCAR Groups) vitamin A and E serum levels increased in the three treated groups compared to control one (p < 0.05). Milk lipoperoxide content was statistically lower in treated cows than in control ones, whereas, as to vitamins, only vitamin E increased in all the treated groups compared to control. Taken together, the results of this study confirm the efficacy of antioxidant administration in the early postpartum of dairy cows and suggest that an excess in antioxidant administration may result, paradoxically, in an increase in ROS generation.

Evaluation of blood and milk oxidative status during early postpartum of dairy cows.

In dairy cows, the intensity of metabolic activity, associated with the negative energy balance (NEBAL), is responsible for an increased production of reactive oxygen species (ROS) and, subsequently, for the development of the condition of oxidative stress, which may overwhelm the antioxidant potential of the bovine maternal organism, making it prone to the development of many puerperal dysfunctions, as well as to an alteration of colostrum and milk quality. Given these premises, the aims of this study are to evaluate serum and milk concentrations of ROS and lipoperoxides, vitamins A and E, on the 10th, 12th, 14th and 16th day postpartum of dairy cows, a particularly critical period during which the NEBAL reaches its nadir, and to compare the trends of these parameters in two different bovine breeds. The study was performed in pluriparous Italian Friesian and Brown dairy cows. On the 10th day postpartum, all cows underwent a clinical examination to exclude the presence of alterations; furthermore, on the same day, a milk sample was collected from each cow, in order to perform the somatic cell count (SCC; (CE) N. 853/2004) and to establish which of them had an SCC ≤400,000/ml or >400,000/ml. In this study, among the 110 cows that were initially selected, the evaluation of these parameters allowed the inclusion of 80 animals, which were divided into four groups of 20 subjects each: Group F and F1: Italian Friesian healthy cows, with SCC ≤400,000/ml and >400,000/ml, respectively; Group B and B1: Italian Brown healthy cows, with SCC ≤400,000/ml and >400,000/ml, respectively. On the 10th, 12th, 14th and 16th day postpartum, peripheral blood and milk samples were collected. The results obtained show that in group B1 there were higher concentrations of ROS and milk antioxidants compared with Friesian group cows. This datum let us suppose that even in the presence of higher ROS concentrations the antioxidant status found in group B1 seems to be able to counteract the oxidative damage, which is more likely to develop in these cows.

Evaluation of serum concentrations of interleukin (IL)-4, IL-10, and IL-12 during pregnancy in bitches.

Complex cytokine networks play an important role in a wide range of pregnancy-related processes. During physiological pregnancy, the balance of T helper lymphocytes Th1 and Th2 is strongly shifted toward Th2, which has a protective role in the feto-maternal interaction. The aim of the present study is to establish the serum concentrations, ranges, and trends of anti-inflammatory interleukin (IL)-4 and IL-10, and inflammatory IL-12A, during three phases of gestation in the bitch (20-30, 31-40, and 41-57 days of gestation). Our results indicate that, in early gestation, IL-4 and -10 serum concentrations are elevated and might depend on progesterone that could act as a potent inhibitor of Th1 responses inducing, conversely, the production of Th2-type cytokines (i.e., IL-10 and IL-4). On the other hand, between 30 and 40 days of gestation, the concentrations of the anti-inflammatory ILs decrease probably because of high concentrations of prolactin, which is endowed with immunostimulatory properties on different immune cell types. In the third phase of gestation (41-57 days), an increase in IL-10 occurs, which might depend on high levels of 17ß-estradiol that, during pregnancy, interfere with the ability of dendritic cells to stimulate T lymphocytes, acting as an anti-inflammatory factor. Conversely, the low and persistent concentrations of inflammatory IL-12A, throughout pregnancy, compared with anti-inflammatory ILs, might depend on the characteristic cytokine products of Thl and Th2 cells that are known to be mutually inhibitory. The monitoring of immunological status via the levels of cytokines during pregnancy in the bitch, could represent a diagnostic tool to predict and/or prevent pregnancy abnormality, as demonstrated in women.

The impact of oxytocin on the hemodynamic features of the milk vein in dairy cows: a Color Doppler investigation.

This manuscript investigates, by Color Doppler ultrasonography, the basal hemodynamic features of the left cranial epigastric vein (milk vein) of 210 Holstein-Friesian cows, throughout lactation. Blood velocity, diameter, blood flow of the milk vein (before and after milking) and milk production detected in cows administered oxytocin were compared with the same parameters found in untreated cows. Blood velocity tended to decrease throughout lactation as did milk production, whereas both diameters and blood flows tended to increase towards the end of the study. All the four parameters showed higher values in the treated cows than in the untreated ones. This study provides a detailed picture of the hemodynamic features of the milk vein of the dairy cows throughout lactation and suggest that oxytocin may exert a vasodilatory effect, in vivo, at least on the cranial epigastric vein of the dairy cow.

The pharmacological pathways of GnRH mediating the inhibition of mammary tumours: implications in humans and domestic animals.

The Gonadotropin-Releasing Hormone (GnRH) exists in two isoforms, GnRH-I and GnRH-II, in most vertebrates, including humans. Both of these isoforms and their respective receptors have been found in many healthy and pathologic extra nervous system tissues, such as cells found in cancers of the reproductive systems and, in particular, in breast cancer. GnRH analogues are used as therapeutic agents in the case of sex-hormone-dependent tumours. Besides acting as suppressors of steroidogenesis, GnRH analogues seem to interfere with mitogenic signal transduction pathways, thus behaving as negative regulators of tumour growth and progression. GnRH analogues counteract the proliferating effects of both epidermal growth factor (EGF) and insulin like growth factor (IGF-I); additionally, it affects the mitogen-activated protein kinase (MAPK) cascade and modulates the activity of the urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitory (PAI) system, which is involved in the process of metastasis. In addition, GnRH analogues decrease the expression of many growth factors involved in the development of human uterine myomas (as well as endometriotic tissue), such as the vascular endothelial growth factor (VEGF), which is deeply implied in the angiogenesis of many benign and malignant tumours, including breast cancer. Angiogenesis is one of the primary processes leading to the progression and metastasis of breast cancer cells, and a key therapeutic goal in the fight against tumours is the blocking of new vessel sprouts. Given these premises, this review provides an update on the background of anti-neoplastic properties of GnRH analogues..

In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts.

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a ß-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a ß-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.

Localization of thyrotropin receptor and thyroglobulin in the bovine corpus luteum.

The receptor of the Thyroid Stimulating Hormone (TSHr) and thyroglobin (TGB), are two proteic factors necessary for the synthesis of hormones, in the thyrocite. In mammals, many immuno-histochemical reports indicate the presence of the TSHr in extra-thyroidal tissues, but not in the ovary. Triiodothyronine (T(3)) and thyroxine (T(4)) have been widely shown to affect ovarian functions and the synthesis of progesterone (P(4)). The aim of this study was to determine if by immunohistochemistry techniques TSHr and TGB could be found in the bovine corpora haemorragica, lutea and albicantia. A primary rabbit polyclonal antibody against human TSHr and a primary rabbit polyclonal antibody against human TGB were employed. Furthermore, the accuracy of bovine thyroid to the antibodies used in this study was tested. A positivity reaction for the anti-TSHr serum in the large luteal cells and immunostaining of both small and large luteal cells with the anti-TGB serum occurred only in mature corpora lutea. No immunostaining was detected in stromal cells, blood and lymphatic vessels and in corpora haemorragica and albicantia. Bovine thyroid tissue showed immunostaining to both the antibodies employed. These data suggest that the luteal cells of mature corpora lutea may be involved in the synthesis of thyroid hormones, which may modulate P(4) synthesis, acting in an autocrine and paracrine way.

Renin and ovarian vascularization in cows with follicular cysts after epidural administration of a GnRH analogue.

The ovarian renin-angiotensin system may play an important role in follicular growth and maturation, as well as in the process of ovulation. The aim of this study was to investigate the effects of administration of a GnRH analogue to cows with ovarian follicular cysts on plasma renin concentrations and ovarian vascularization. This study was performed with 60 Friesian cows, which were diagnosed with follicular cysts, and randomly allocated into two groups: group A (treatment; n=30) received 2ml of lecirelin (Dalmarelin((R)) - Fatro), per head via sacro-coccygeal epidural, and group B (control; n=30) received 2ml saline solution (0.9% NaCl) per head by the same route. Blood samples were immediately collected prior to administration (T0) and then 24h (T1), 48h (T2) and 8 days (T3) after administration of the treatment, for both groups. Ovarian vascularization was evaluated utilizing Power Doppler on these same days in 10 animals from each group. The number of pixels detected by Power Doppler was used as an indicator of the degree of vascularization. Plasma renin concentrations remained relatively constant for the control (group B) animals, but increased as the sampling period progressed (NS) for the treated cows (group A). Similarly, there were no changes in ovarian vascularization (number of pixels) for the control cows, but vascularization increased throughout the sampling period in the treated animals. The number of pixels associated with cysts was significantly higher for treated compared to control cows at 24h after treatment (P<0.001). The epidural administration of a GnRH analogue was determined to be a highly effective therapy for follicular cysts (regression occurred in 82% of treated cows within 8+/-2 days after treatment, but in none of the control cows), which also enhanced ovarian vascularization.

Influence of antibiotic therapy on serum levels of reactive oxygen species in ovariectomized bitches.

This study was conducted on 60 ovariectomized bitches. The objectives were to measure the mean reactive oxygen species (ROS) concentrations before, during and after surgery, and to investigate the effect of the administration of five different antibiotic treatments: amoxicillin, benzylpenicillin/dihydrostreptomycin, sulfametazine/sulfamerazine/sulfathiazole, enrofloxacin, lincomycin/spectinomycin. The first value recorded represented the mean ROS concentration in anestral bitches and constitutes a reference level with which to compare the subsequent measurements. After premedication, induction of anesthesia and during maintenance and surgery, ROS serum concentrations showed constant values until the end of surgery. After surgery and during antibiotic administration, an increase in ROS concentration occurred, which differed among the five groups in relation to the antibiotics employed. The lowest increases occurred in the groups treated with the combination of lincomycin/spectinomycin, and with amoxicillin; whereas the highest increases were detected in the group treated with enrofloxacin. The three other antibiotics showed an intermediate level of influence on oxidative status.