Levels of selected cytokines and acute-phase proteins in the serum of dairy cows with cystic ovarian disease and those in follicular and luteal phases of normal ovarian cycle.

The aim of the study was to evaluate the concentration of selected cytokines (tumor necrosis factor [TNFα], interleukin [IL]-6, IL-10) and acute-phase proteins (APPs; haptoglobin [Hp] and serum amyloid A [SAA]) in the serum of cows with follicular and luteal ovarian cysts and in those in the follicular and luteal phases of a physiological ovarian cycle. The study was conducted on 24 Holstein-Friesian (HF) cows: eight cows in the follicular phase of the cycle; the same cows after 10 days when they were in the luteal phase; eight cows with follicular cysts; and eight cows with luteal cysts. Levels of progesterone, 17ß-estradiol, TNF-α, IL-6, IL-10, SAA, and Hp were evaluated in the serum of all examined cows. The concentration of the assessed parameters in the serum was determined using commercially available bovine ELISA kits that were specific for each parameter. The results obtained then showed that values of TNF-α and IL-6 were highest in cows with luteal cysts (p < .001), compared to those with other ovarian structures. The highest level of IL-10 was recorded in cows with two types of ovarian cysts. Furthermore, high values of SAA and Hp were found in cows with two types of cysts; however, these values were higher in cows with follicular cysts. The present study shows that an assessment of the levels of cytokines (pro- and anti-inflammatory) and APPs in the serum may be important in the investigation of the processes underlying the formation and differentiation of ovarian cysts in dairy cows.

Serum amyloid A protein (SAA), haptoglobin (Hp) and selected hematological and biochemical parameters in wild mares before and after parturition.

The aim of the study was to evaluate physiological changes in hematological and biochemical parameters in mares in perinatal period. Blood samples were collected from 24 pregnant Polish Konik breed mares which were divided into two groups. The first group (Group - I, n=12) comprised mares living in the wild, in the reserve. The second group (Group - II, n=12) consisted of mares kept in stables. The blood was collected 2 weeks prior to the parturition, then 24 hours after the delivery, and then at the 7th and 21st day after foaling. When comparing the two groups before the parturition, no significant differences in terms of WBC, RBC, and Hb were found, however, there was a significant difference in MCV, MCH, LYM, NEU and SEG NEU (p≤0.05). In Group II, 24 hours after the parturition and at the 21st day after foaling, a significant raise in WBC, NEU and SEG NEU (p≤0.05) was detected. No significant differences in serum concentrations of proteins such as TP, Alb or Glb were observed. As to acute phase proteins, significant rise in SAA and Hp (p≤0.05) was found in the two examined groups 24 hours after the parturition. Yet, this rise remained within physiological range. The study revealed a certain degree of fluctuations in hematological parameters, in serum concentrations of acute-phase proteins and total proteins in the mares in the perinatal period. However, these changes remained still within physiological ranges and thus they do not indicate potential susceptibility to disorders of perinatal period.

Sperm quality and selected biochemical parameters of seminal fluid in dogs with benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) in dogs is most commonly associated with age and increasing concentrations of dihydrotesterone, a hormone that stimulates growth and secretion of the prostatic epithelial cells. During this process, the biochemical composition of prostatic secretion changes, which can affect the quality of semen and limit the ability of the sperm to contribute to fertilization. Therefore, the present study was conducted to examine possible correlation between BPH and biological quality of semen. The study was performed in 11 sexually mature dogs of various breeds. Animals were divided into two groups: healthy dogs (Group I; n = 5; mean age 4.32; SEM = 1.28) and dogs with BPH (Group II n = 6; mean age 6.16; SEM = 0.65). Semen and prostate secretions were collected and evaluated in this study. Standard semen examinations were conducted in the ejaculates collected; moreover, the extent of apoptosis and DNA defragmentation was determined. The selected biochemical parameters were determined in the prostate secretion. According to the examination results, there were no significant differences in standard semen parameters between the two groups of dogs. Nevertheless, morphological tests of semen in dogs with BPH demonstrated elevated percentages of primary defects in spermatozoa. A significant increase (P = 0.01) in DNA defragmentation of sperm was found in dogs with BPH. Moreover, changes in the biochemical composition of prostate secretion were demonstrated. In dogs with BPH, pH of prostate secretions was greater (P = 0.03), concentrations of cholesterol increased while concentrations of Zn and Cu decreased. The study findings reveal that BPH does not change semen quality in dogs.

Flow cytometric evaluation of sperm apoptosis in semen of silver foxes in the breeding period.

The objective of the study was to evaluate cytometrically the percentage of apoptotic and necrotic spermatozoa in fresh semen of silver foxes in the breeding season. In males F3 and F4 with high percentages of early apoptotic (A+Pi-), late apoptotic (A+Pi+) and necrotic (A-Pi+) spermatozoa as well as 56-65% of living spermatozoa (A-Pi-) with progressive motility, the semen was characterised by reduced fertility. In males F1 and F2 with spermatozoa showing the motility and viability of 89-90% and high percentages of living cells that do not bind Annexin V and propidium iodide, the semen was assessed as valuable and useful for artificial insemination. Amongst 16 females of group I and II inseminated with semen from F1 and F2 males, 15 (93.75%) had multi-cub litters - on average 6.1 and 4.8, respectively. In contrast, amongst 16 females of group III and IV inseminated with semen from F3 and F4 males, only 10 (62.5%) had litters with few cubs (on average 2.6 in group III and 2.1 in group IV). Our findings explicitly indicate that semen of farm male foxes should be evaluated before the breeding season, as one of the causes of reproduction failures is likely to be a high percentage of apoptotic and necrotic spermatozoa. Thanks to flow cytometry, fresh ejaculates can be speedily evaluated and their usefulness for artificial insemination determined.

Assessment of extent of apoptosis and DNA defragmentation in chilled semen of stallions during the breeding season.

The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. The results of basic tests did not demonstrate significant differences amongst stallions, except for progressive sperm motility, which was significantly higher (p < 0.05) in the semen of Arabian stallions. In the semen of the same stallions, a significant decrease in the percentage of alive spermatozoa was observed at 72, 96 and 120 h of storage, whereas a significant increase in the number of spermatozoa with DNA defragmentation was found after 24 h storage. In the semen of coldblood stallions, significantly reduced live spermatozoa percentage was observed at 96 and 120 h, while increased DNA defragmentation was observed at 48 h. These findings demonstrated that the semen of Arabian stallions chilled to +4°C retained original characteristics until 24 h of storage, whereas in coldbloods, these were preserved up to 48 h of storage.

Serum levels of acute phase proteins: SAA, Hp and progesterone (P4) in mares with early embryonic death.

The study involved 46 healthy purebred Arabian mares exhibiting regular oestrous cycles that underwent artificial insemination (AI). Pregnancy was detected ultrasonographically (US) in 40 mares. In 15 mares in foal, early embryonic death (EED) was observed during the pregnancy days 14-21. Blood for determinations of serum acute phase proteins (SAA and Hp) and progesterone (P4) was sampled 12-24 h before ovulation and the first insemination, at 12, 24, 72, 96 h and on day 7, 10, 14, 21, 35 and 55 after ovulation. The results revealed that in 25 mares without EED, the serum levels of P4, SAA and Hp were within physiological limits; in 15 mares with EED, the levels of SAA and Hp were significantly increased. In seven mares with EED, high levels of SAA and Hp were already found before ovulation and at 12, 24, 72, 96 h as well as on day 7 and 10 post-ovulation, whereas the level of P4 was normal for early pregnancy. In the remaining eight mares with EED, increased levels of SAA and Hp were found at 72 h after ovulation and maintained until day 55. In this group, the level of P4 decreased since 96 h after ovulation. Determinations of SAA, Hp and P4 in mares in early pregnancy (EP) are useful for monitoring normal development of pregnancy and for diagnosis of subclinical genital inflammations, which may lead to EED.

Usefulness of C-reactive protein, serum amyloid A component, and haptoglobin determinations in bitches with pyometra for monitoring early post-ovariohysterectomy complications.

The aim of this study was to assess changes in serum concentrations of C-reactive protein (CRP), serum amyloid A component (SAA), and haptoglobin (Hp) in bitches with pyometra undergoing ovariohysterectomy that developed postoperative wound infection-related complications. The study revealed that difficulties in postoperative wound healing were induced by infections with Escherichia coli and Staphylococcus spp. leading to re-increased levels of CRP and SAA immediately after surgery and persistently high Hp levels throughout the experiment. Our results indicate that acute-phase proteins in bitches undergoing surgery because of pyometra are useful markers for monitoring the postoperative period. Moreover, they enable prompt therapeutic management once complications develop.

The effect of nonspecific immunostimulation of pregnant mares with 1,3/1,6 glucan and levamisole on the immunoglobulins levels in colostrum, selected indices of nonspecific cellular and humoral immunity in foals in neonatal and postnatal period.

The objectives of the studies were to evaluate the effect of levamisole and 1,3/1,6 glucan applied in pregnant mares on parameters of non-specific cellular and humoral immunity of foals. Eighteen mares in three experimental groups (six animals in each) and their progeny were examined. Multiparous mares, crossbreed of Polish, full-blood and Hannover lines (400-500 kg), 4-9 years old, originated from four different farms. They were kept under identical zoohygienic and nutritional conditions. The animals were randomly chosen in experimental groups. None of mares had been previously vaccinated. In group I, levamisole was injected three times at 7-day intervals at a dose of 2.5 mg/kg of body weight. Group II was injected at the same periods of time and manner with 1,3/1,6 glucan at a dose of 0.19 mg/kg of body weight, whereas mares in group III served as controls. Injection of the immunostimulators started in mares 4-6 weeks before expected parturition. Blood was taken from foals before the first dose of colostrum, then 18 and 36 h after the first dose of colostrum and on Days 7, 14, 21, 28, 35, 42, 49 and 56 of life. The parameters determined in blood were reduction of NBT by PMNs, phagocytic activity, phagocytic index, test of intracellular killing and in blood sera were total protein, gamma-globulin fraction, lysozyme activity, level of IgG, IgG(T), IgM and IgA. In the first dose of colostrum taken just after parturition, specific gravity, total protein, gamma-globulin complex, lysozyme activity, level of IgG, IgG(T), IgM, IgA were determined. Colostrum of mares immunostimulated with levamisole or 1,3/1,6 glucan were characterized by a high content of IgG and IgG(T) compared to the colostrum of nonstimulated mares. The level of immunity was higher in foals from dams immunostimulated with levamisole or 1,3/1,6 glucan. Clinical examinations in neonatal and postnatal period did not show any abnormalities in these foals.

The Genome Sequence DataBase (GSDB): improving data quality and data access.

In 1997 the primary focus of the Genome Sequence DataBase (GSDB; www. ncgr.org/gsdb ) located at the National Center for Genome Resources was to improve data quality and accessibility. Efforts to increase the quality of data within the database included two major projects; one to identify and remove all vector contamination from sequences in the database and one to create premier sequence sets (including both alignments and discontiguous sequences). Data accessibility was improved during the course of the last year in several ways. First, a graphical database sequence viewer was made available to researchers. Second, an update process was implemented for the web-based query tool, Maestro. Third, a web-based tool, Excerpt, was developed to retrieve selected regions of any sequence in the database. And lastly, a GSDB flatfile that contains annotation unique to GSDB (e.g., sequence analysis and alignment data) was developed. Additionally, the GSDB web site provides a tool for the detection of matrix attachment regions (MARs), which can be used to identify regions of high coding potential. The ultimate goal of this work is to make GSDB a more useful resource for genomic comparison studies and gene level studies by improving data quality and by providing data access capabilities that are consistent with the needs of both types of studies.

Rapid DNA sequencing based upon single molecule detection.

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.