The Potential of Sheep or Camel Milk Constituents to Contribute to Novel Dressings for Diabetic Wounds.

Impaired wound healing is a complication of diabetes, which constitutes a serious problem in clinical practice. Currently, there is a high demand on the market for local treatment options for difficult-to-heal wounds caused by diabetes. The development of dressings that accelerate wound healing has recently been the subject of much research. Sheep and camel milk is gaining importance due to the content of many bioactive substances with health-promoting effects, such as insulin, LF, proline, or CLA. Sheep and camel milk proteins are a promising source of insulin, antidiabetic, and antihypertensive peptides. Numerous studies show that local administration of insulin has a significant impact on the healing of diabetic wounds. Sheep and camel milk, due to the highest LF content among ruminants, reduces autoimmune inflammatory processes and protects against bacterial and viral infections in the wound environment. Sheep's milk has the highest content of proline and CLA, and their addition to a hydrogel dressing can help in the development of an effective dressing material. The production of hydrogel dressings containing sheep and camel milk, which are naturally rich in the bioactive substances presented in this review, may be a promising step in the market of specialized dressings for difficult-to-heal diabetic wounds.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells.

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10-250 µM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 µM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

The Improved Method for Determination of Orotic Acid in Milk by Ultra-Fast Liquid Chromatography with Optimized Photodiode Array Detection.

Ultra-fast liquid chromatography (UFLC) with a photodiode array detector (DAD) for simple and rapid determination of orotic acid (OAc) in milk of sheep and cows is described. Milk samples are treated with acetonitrile (1:1, v/v) and then centrifuged at 4 °C. To 1 mL of the obtained supernatant 9 mL of ultrapure water was added. Subsequently, 0.5-6 µL of the resulting solution was injected into the UFLC-DAD system. Separation and quantification of OAc in milk samples was achieved using two Kinetex C18 columns (1.7 µm, 150 mm × 2.1 mm, i.d., 100 Å; Phenomenex) fitted with a pre-column of 4 mm × 2 mm, i.d. (Phenomenex) containing C18 packing material. All separations were performed at a column temperature of 35 °C while the ambient temperature was 21-24 °C. Satisfactory separation of OAc from endogenous species of milk can be achieved using the binary gradient elution program and UV detection at wavelengths 278 nm. Our original procedure resulted in suitable separation and quantification of OAc in milk samples; OAc eluted at 6.44 ± 0.03 min. The total run time of OAc analysis (including re-equilibration) was 27 min. As expected, the OAc peak was absent from the blank when the proposed gradient elution program and UV detection at 278 nm was used. The average recoveries of OAc standards added to milk samples were satisfactory (96.7-105.3%). The low inter-and intra-assay coefficient of variation derived from the measurements of OAc in cow and ovine milk samples (i.e., 0.784%, 1.283% and 0.710%, 1.221%, respectively) and in O-Ac standards (i.e., 0.377% and 0.294%, respectively), as well as high recoveries of OAc added to ovine and cows' milk (~100%) and the low detection (0.04 ng) and quantification (0.12 ng) limits point to satisfactory accuracy, precision and sensitivity of the reported method. OAc concentrations in ovine milk samples were within the range from 25 to 36 mg/L, while OAc levels in cows' milk samples was found in the range of 32-36 mg/L. Our original procedure is suitable for routine quantification of OAc in milk of ewes and cows.

Importance of Bioactive Substances in Sheep's Milk in Human Health.

Sheep's milk is an important source of bioactive substances that have health-promoting functions for the body. The valuable composition of sheep's milk is due to the high content of fatty acids, immunoglobulins, proteins, hormones, vitamins and minerals. Many biopeptides found in milk have antibacterial, antiviral and anti-inflammatory properties. The bioactive substances of sheep's milk also show anticancer properties. Sheep's milk, thanks to its content of CLA and orotic acid, prevents the occurrence of type 2 diabetes, Alzheimer's disease and cancer. Sheep's milk, as a product rich in bioactive substances, can be used as a medical aid to support the body in the fight against neurological and cancer diseases.

Impact of Photoperiod Length and Treatment with Exogenous Melatonin during Pregnancy on Chemical Composition of Sheep's Milk.

The aim of the study was to determine the effect of photoperiod and exogenous melatonin on milk yield and chemical composition of sheep's milk. Sheep (n = 60) were randomly divided into three groups: lambing in February (Group 1-n = 20), lambing in June (Group 2-n = 20), and lambing in June and treated with subcutaneous melatonin implants (Group 3-n = 20). Milk yield was higher for Group 1 and Group 2 than for Group 3 (p < 0.01). The milk of ewes of Groups 2 and 3 had a significantly (p < 0.01) higher content of dry matter, protein, and fat. Group 3 sheep's milk contained significantly more (p < 0.01) of SFA (Saturated Fatty Acids). The highest content of MUFA (Monounsaturated Fatty Acids) and PUFA (Polyunsaturated Fatty Acids) was found in the samples collected from Group 1, the lowest was in the milk of Group 3 animals. The highest (p < 0.01) CLA, content was identified in the milk of Group 1, while the lowest was recorded for the milk obtained from sheep treated with exogenous melatonin (Group 3). The experiment carried out has shown that day length and treatment with exogenous melatonin modulate the chemical composition of milk.

Involvement of orexin A in nocturnal melatonin secretion into the cerebrospinal fluid and the blood plasma in seasonal sheep.

In sheep, differences in orexin A (OXA) gene expression and activity are related to changes in energy demand and seasonal reproduction. However, the mechanism by which and the key place where the OXA signal is integrated with photoperiod, whose main biochemical expression is melatonin (MEL), remain unknown. We examined the effects of cisterna magna injections of OXA (0.3 µg/kg body weight) on nocturnal cerebrospinal fluid (CSF) and plasma MEL concentrations; mRNA and protein expression of two rate-limiting enzymes for MEL biosynthesis, tryptophan 5-hydroxylase-1 (TPH1) and arylalkylamine-N-acetyltransferase (AA-NAT); and OXA receptor (OX1R, OX2R) expression in the pineal gland (PG) obtained from twenty ewes during the short-day (SD) and long-day (LD) seasons. OXA increased (P < 0.001) CSF and plasma MEL concentrations regardless of the season. Plasma MEL was positively correlated (P < 0.001) with CSF MEL in the OXA-treated sheep in both seasons. OXA had no effect (P > 0.05) on TPH1 transcript or protein level but upregulated (P < 0.05) AA-NAT mRNA and protein expression in both seasons. OXA enhanced (P < 0.05) OX1R mRNA level only during the LD season. Our results show that the endocrine activity of the ovine PG is regulated by day length and non-photic signals via hypothalamic OXA. These results are important for understanding the work of the biological clock and recognizing mechanisms responsible for the adaptation of seasonal animals to the changing external environment conditions. OXA and MEL are both involved in the regulation of the sleep-wakefulness system, therefore our results can be used in the study on the circadian rhythm disorders in humans (e.g. jet lag, insomnia, seasonal depression).

The effects of leptin on plasma concentrations of prolactin, growth hormone, and melatonin vary depending on the stage of pregnancy in sheep.

The effects of hyperleptinemia and leptin resistance during gestation are unclear. Leptin, an important neuroendocrine regulator, has anorexic effects, but its interactions with other metabolic hormones during pregnancy are unclear. We examined potential roles of leptin in regulating prolactin (PRL), GH, and melatonin plasma concentrations during pregnancy in Polish Longwool ewes. Twelve estrus-synchronized ewes carrying twins after mating were randomly assigned to receive i.v. injections of saline or recombinant ovine leptin (2.5 or 5.0 µg/kg BW). Blood samples were collected (15-min intervals over 4 h) immediately before the first injection at dusk and kept under red light. Treatments were repeated at 2-wk intervals, starting before mating and continuing from days 30 to 135 of gestation. Concentrations of plasma PRL, GH, and melatonin were determined using a validated RIA. The effects of leptin on hormone plasma concentrations varied depending on pregnancy stage and leptin dose. PRL plasma concentrations were affected at most stages of pregnancy and before gestation. In non-, very early- (day 30), and late- (day 120 and 135) pregnant ewes, exogenous leptin stimulated PRL (P < 0.001) plasma concentrations, while during the second month of gestation, it decreased PRL concentrations (P < 0.01). Leptin affected GH plasma concentrations (P < 0.05) only during the first 2 mo of pregnancy, with no effects during the second part of gestation or before pregnancy. In early-pregnant ewes (day 30 and 45), leptin decreased melatonin plasma concentrations (P < 0.05), but at day 60, leptin stimulated melatonin plasma concentrations at low (P < 0.01) and high doses (P < 0.05), with no effects in ewes after 105 d of gestation. These data indicate specific pregnancy-induced endocrine adaptations to changes in energy homeostasis, supporting the hypothesis that leptin affects PRL, GH, and melatonin release during gestation.

Effects of salsolinol and its antagonistic analogue, 1-MeDIQ, on growth hormone release in nursing sheep.

Suckling induces a GH surge simultaneously to that of prolactin, so we tested whether salsolinol, a dopamine derivative (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), participates in the regulatory process of GH secretion in lactating sheep. A series of intracerebroventricular (i.c.v.) infusions of salsolinol, in two doses, was performed in nursing sheep, without suckling, during the fifth week of lactation. In other suckling sheep, we infused i.c.v. a structural analogue of salsolinol-1-methyl-3,4-dihydroisoqinoline (1-MeDIQ), which is able to antagonize salsolinol's action. Intracerebroventricular treatment of nursing sheep with a lower dose of salsolinol (total 50 ng) significantly increased plasma GH concentration, as compared with the concentrations noted before the infusion and in nursing controls. A higher dose of salsolinol (total 5 micrograms) did not affect GH release significantly. Intracerebroventricular treatment with 1-MeDIQ (total 300 micrograms) significantly reduced basal GH release, not affecting a pattern of GH surge in response to suckling. In conclusion, salsolinol may affect the regulatory process of GH secretion in lactating sheep, but its role seems not to be major.

Identification of salsolinol in the mediobasal hypothalamus of lactating ewes and its relation to suckling-induced prolactin and GH release.

The push-pull perfusions of the infundibular nucleus-median eminence (IN/ME) were made in lactating ewes (n=7) twice, to identify dopamine (DA)-derived salsolinol and the changes in its extracellular concentration in response to suckling. The perfusate collecting period in every ewe consisted of control non-suckling period, 1000-1230 h (five perfusates), and suckling period, 1230-1500 h (next five perfusates). Simultaneously, blood samples were collected from 1000 to 1500 h at 10-min intervals. The perfusate concentrations of salsolinol and DA were measured by HPLC, and plasma prolactin and GH concentrations were assayed by the RIA. Mean concentrations of salsolinol in perfusates collected from the anterior and posterior parts of the IN/ME (according to post-mortem localization of a perfusion site) increased significantly (P<0.05 and P<0.001 respectively) during the suckling period, when compared with those noted during the non-suckling period. While no DA was found in the anterior part, only vestigial amounts of DA were found in a few perfusates collected from the posterior part. Salsolinol was not detected in the IN/ME of ewes 10 weeks after weaning (seasonal anoestrus). Mean plasma prolactin and GH concentrations during suckling were significantly (P<0.001) higher than those noted during the non-suckling period. In conclusion, our current study reveals that salsolinol is present in the IN/ME of lactating ewes and that its extracellular concentration increases during suckling. Moreover, it supports the role of salsolinol as a neurotransmitter involved in the regulatory process of prolactin secretion at least during lactation.

The effect of long term exposure to copper on physiological condition and reproduction of sheep.

The influence of copper upon some physiological parameters and reproduction in ewes was studied. Four groups of animals were investigated: 1/ control ewes (untreated); 2) ewes receiving copper as a supplement over the recommended amount of copper in food (10, 25 or 50 mg Cu/ewe/day); 3/ control, superovulated ewes; and 4) ewes treated with 50 mg copper during one month and then superovulated. After 10 months of daily exposure to 10 mg of copper/ewe/day no signs of toxicity on physiological condition and reproduction were found. In ewes exposed to 25 or 50 mg of copper a decrease in blood parameters and increase in concentration of Cu in blood and liver were noticed. The wavy pattern of follicles was disturbed and disorders in fecundity, prolificacy and pregnancy occurred. Significant differences between the number of corpora lutea in superovulated control animals and experimental (Cu 50 mg) ewes were observed.