R. Rajmon

Influence of organic versus inorganic dietary selenium supplementation on the concentration of selenium in colostrum, milk and blood of beef cows

BackgroundSelenium (Se) is important for the postnatal development of the calf. In the first weeks of life, milk is the only source of Se for the calf and insufficient level of Se in the milk may lead to Se deficiency. Maternal Se supplementation is used to prevent this.We investigated the effect of dietary Se-enriched yeast (SY) or sodium selenite (SS) supplements on selected blood parameters and on Se concentrations in the blood, colostrum, and milk of Se-deficient Charolais cows.MethodsCows in late pregnancy received a mineral premix with Se (SS or SY, 50 mg Se per kg premix) or without Se (control – C). Supplementation was initiated 6 weeks before expected calving. Blood and colostrum samples were taken from the cows that had just calved (Colostral period). Additional samples were taken around 2 weeks (milk) and 5 weeks (milk and blood) after calving corresponding to Se supplementation for 6 and 12 weeks, respectively (Lactation period) for Se, biochemical and haematological analyses.ResultsColostral period. Se concentrations in whole blood and colostrum on day 1 post partum and in colostrum on day 3 post partum were 93.0, 72.9, and 47.5 μg/L in the SY group; 68.0, 56.0 and 18.8 μg/L in the SS group; and 35.1, 27.3 and 10.5 μg/L in the C group, respectively. Differences among all the groups were significant (P < 0.01) at each sampling, just as the colostrum Se content decreases were from day 1 to day 3 in each group. The relatively smallest decrease in colostrum Se concentration was found in the SY group (P < 0.01).Lactation period. The mean Se concentrations in milk in weeks 6 and 12 of supplementation were 20.4 and 19.6 μg/L in the SY group, 8.3 and 11.9 μg/L in the SS group, and 6.9 and 6.6 μg/L in the C group, respectively. The values only differed significantly in the SS group (P < 0.05). The Se concentrations in the blood were similar to those of cows examined on the day of calving. The levels of glutathione peroxidase (GSH-Px) activity were 364.70, 283.82 and 187.46 μkat/L in the SY, SS, and C groups, respectively. This was the only significantly variable biochemical and haematological parameter.ConclusionSe-enriched yeast was much more effective than sodium selenite in increasing the concentration of Se in the blood, colostrum and milk, as well as the GSH-Px activity.

Nitric oxide-dependent activation of pig oocytes: role of calcium.

Pig oocytes matured in vitro are parthenogenetically activated after treatment with nitric oxide (NO)-donor SNAP. The chelation of intracellular calcium ions with BAPTA-AM suppressed the SNAP-induced activation in a dose-dependent manner. Activation by a NO-donor is dependent on the influx of calcium from extracellular spaces, because the blockage of calcium channels by verapamil had significantly reduced the activation rate in SNAP-treated oocytes. The blockage of inositol triphosphate receptors had no effect on the activation of oocytes by a NO-donor. On the other hand, the blockers of ryanodine receptors, procaine and ruthenium red, inhibited the activation of oocytes induced by a NO-donor. These data indicate that the activation of pig oocytes by a NO-donor is calcium-dependent. The calcium for the activation is mobilized from extracellular and intracellular spaces. For the mobilization of intracellular calcium stores, it is the ryanodine receptors and not the inositol triphosphate receptors that play a key role.

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway.

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.

Experimental study of non-alcoholic fatty liver disease (NAFLD) on a model of starving chickens: Is generalization of steatosis accompanied by fibrosis of the liver tissue?

The objective of this work was to study the mechanism of liver parenchyma development under the influence of restriction of diet. Useful information is presented about the pathologic features associated with diet restriction in a chicken animal model of NAFLD. There were 96 chickens of two genotypes, Ross 308 and Cobb 500, in the experiment. The control group was fed a standard mixture ad libitum (ADL). The first experimental group, under restriction from the age of 2 weeks, was fed 80% ADL. The second experimental group was fed 65% ADL from the age of 2 weeks. There were 16 animals in each group. The experiment lasted 5 weeks. Liver parenchyma samples were obtained at the age of 35 days by the necropsy method and then processed by standard histologic methods. The slices were stained by standard staining: hematoxylin-eosin and by Sirius red kit for collagen type I and reticulin visualization. Hepatocyte diameter and the proportion of interstitial tissue to the parenchyma of the liver were measured objectively. Microvesicular liver steatosis was observed after 35 days of restriction. Hepatocyte diameter was significantly influenced by sex, genotype, and the experimental group. The proportion of interstitial tissue to the liver parenchyma was highly influenced by genotype and group, but there were no interactions. An increase in the steatosis histologic grade is associated with inflammatory changes, with decrease of hepatocyte diameter and with a decreasing proportion of interstitial tissue to the liver parenchyma. The results show that early restriction is not associated with the development of fibrosis of the liver tissue.

Adverse effect of tetracycline and doxycycline on testicular tissue and sperm parameters in CD1 outbred mice

Tetracycline and doxycycline are commonly used antibiotics in acne treatment during puberty in humans. The long-term effect of these antibiotics on male reproductive tract development has not been fully elucidated. For this reason we tested the effect of antibiotics on the reproductive parameters of mice males during puberty with the therapeutic dose used in humans, and with lower and higher doses. The outbred mouse strain CD1 with higher heterozygosity was exposed for 14 days at puberty. Adult males at the age of 70 days were used for the measurements. We observed a significant decrease in anogenital distance and thickness of the seminiferous epithelium in the treated animals. Pathological changes in the testes had an impact on sperm quality; a higher number of sperm positively stained with Annexin V and TUNEL and a lower number of acrosome-intact sperm was detected. In conclusion, the treatment of male mice with antibiotics in puberty led to long-lasting effects on reproductive organs and spermatozoa in adult males.

Effect of proteasome inhibitor MG132 on in vitro maturation of pig oocytes.

The present study aimed to demonstrate the dependence of meiotic maturation in pig oocytes on the activity of the protease complex proteasome. The proteasome inhibitor MG132 blocked the exit of maturing pig oocytes from metaphase I stage. Seventy-five per cent of the oocytes were blocked at metaphase I when they were cultured with 10 microM MG132. The blocking effect of MG132 was expressed only when the oocytes were exposed to an inhibitor before the 18th hour of in vitro culture. The effects of MG132 are fully reversible. However, a significant proportion of oocytes (46%) cultured for 48 h in MG132-supplemented medium and then for 24 h in MG132-free medium did not block meiosis at the stage of metaphase II and underwent spontaneous parthenogenetic activation. On the basis of our data we can conclude that exit from the metaphase I stage of meiosis is proteasome-dependent in pig oocytes matured in vitro. On the other hand, our data also indicate that other proteasome-independent events are involved in regulating the exit from metaphase I.

Ultrastructural localisation of calcium deposits in pig oocytes maturing in vitro : effects of verapamil

The culture of pig oocytes in the presence of the calcium channel blocker verapamil (0.02 mM) resulted in the blocking of meiosis at the metaphase I stage, and only a small fraction (about 28%) of the oocytes were able to continue their maturation to the stage of metaphase II. Hence, meiotic maturation in pig oocytes is a calcium-dependent process. After isolation of the pig oocytes from their follicles, the intracellular calcium deposits in the oocyte and granulosa cells, detectable using the combined oxalate-pyroantimonate method, are depleted. The amount of calcium deposits in the oocyte and granulosa cells increased during oocyte meiotic maturation in vitro, especially in the nucleus, mitochondria, vacuoles and cytoplasm. The replenishment of calcium deposits is significantly changed under the effect of verapamil. The increase in calcium deposits in the oocyte nucleus was delayed, a much larger amount of deposits was formed in the mitochondria, and the amount of deposits in the vacuoles was demonstrably smaller. A significant peak in the accumulation of calcium deposits was observed in the cytoplasm of verapamil-treated oocytes after 16 h of in vitro culture. We propose that an altered pattern in the replenishment of calcium deposits can disturb intracellular signalling and prevent the exit of oocytes from the metaphase I stage.

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro.

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).

Nitric oxide and meiotic competence of porcine oocytes.

Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor l-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.

Ultrastructural localisation of calcium deposits in the mouse ovary

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate-pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.