Effect of Rooster Macrophages on the Motility Characteristics of Spermatozoa

The aim of our study was to detect macrophages in rooster semen and subsequently to asses their effect on the spermatozoa motility parameters. Semen samples were collected from Ross PM3 breeder males (n = 24) by the dorso-abdominal massage into prepared sterile tube. Macrophages were identified using fluorescent dye Alexa Fluor-AcLDL and evaluated under a Leica fluorescent microscope (Leica Microsystem, Germany). Roosters (n=8) with the occurrence of macrophages above 20% per samples were classified to the group R1 and roosters (n=16) with the occurrence of macrophages 0-10% were classified to the group R2. The mean content of macrophages was 23.43±1.82 vs. 7.59±1.39 (group 1 vs. group 2, P 5 µm/s) and percentage of progressive motile spermatozoa (motility > 20 µm/s) of two heterospermic samples were measured using computer-assisted sperm analysis (CASA system, Sperm Vision™). We observed significantly (P<0.05) lower progressive motile of rooster spermatozoa in the group R1 in comparison to group R2 (25.04±5.98 vs. 46.38±3.28). Based on the observed preliminary results, we hypothesized that the higher presence of macrophages in semen may have a negative effect on the quality of rooster spermatozoa in terms of their motility. Further experiments are required in order to prove our hypothesis

Comparison of semen characteristics and histological structure of the testis from transgenic and non-transgenic rabbits

[EN] The aim of this study was to compare semen characteristics including sperm quantity, quality, and abnormalities, as well as histological structure of the testis of three-year old transgenic (human clotting factor, hFVIII, gene) and nontransgenic rabbits. For the experiment, 10 transgenic rabbits of F2 and F3 generations and 10 randomly selected non-transgenic males of the same breed and age were used as controls. All males were housed in individual cages, under a the same environmental conditions: photoperiod (14L:10D), temperature (18-20°C), and humidity (65-70%). Semen samples, collected once a week for 20 wk from each control and transgenic male, were analyzed by computer assited semen analysis within a few minutes following natural ejaculation into an artificial vagina. Concentration of spermatozoa was higher in the transgenic than in the non-transgenic group (P<0.001; 316.6±148.8 and 126.7±64.4¿106/ mL, respectively). Significant differences (P<0.1) between transgenic and non-transgenic males were observed also in spermatozoa motility (63.08 vs. 32.60%). Significantly higher (P<0.05) relative volume (8.08±2.89%) and diameter of testicular lumen (36.89±23.11 ¿m) were found in the transgenic animals compared to control animals (16.69±4.70%, 53.89±25.42 ¿m). Our results show that spermatozoa parameters and histological structure of the testis can be used for the characterization of male reproductive traits of older transgenic rabbits.This work was supported by the grant No: 2003 SP51/028 09 00/028 09 03 coordinated by the Slovak Academy of Science and by the grant of Ministry of Agriculture Slovak Republic (RÚVV 07-012). All experiments were approved according to ethical permission No. SK P 28004. We are grateful to Dr. Shoubadeep Roychoudhury for the English correction.Lukac, N.; Massanyi, P.; Flesarova, S.; Danko, J.; Makarevich, A.; Chrenek, P. (2009). Comparison of semen characteristics and histological structure of the testis from transgenic and non-transgenic rabbits. World Rabbit Science. 17(4):221-226. https://doi.org/10.4995/wrs.2009.64722122617

MultiPriDe: automated batch development of quantitative real-time PCR primers

Quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) is a commonly employed gene expression quantification technique. This requires the development of appropriately targeted oligonucleotide primers, which necessitates the identification of ideal amplicons, development of optimized oligonucleotide sequences under most favorable pre-determined reaction conditions, and management of the resultant target-oligonucleotide pair information for each gene to be studied. The Primer3 utility exists for development of oligonucleotide primers and fills that role effectively. However, the manual process of identifying target sites and individually generating primers is inefficient and prone to user-introduced error, especially when a large number of genes are to be examined. We have developed MultiPriDe (Multiple Primer Design), a Perl utility that accepts batch lists of Gene database identifiers, collects available intron and exon position data critical to qRT–PCR primer development, and supplies these sites as identified targets for the Primer3 utility. This automated ‘gene to primer’ procedure is coupled with a set of optimized hybridization conditions used by the Primer3 utility to maximize successful primer design. MultiPriDe and assembled repeat libraries are available upon request. Please direct requests to [email protected]

The camp analogue, dbcAMP can stimulate rabbit reproductive functions: I. Effect on ovarian folliculogenesis, ovulation and embryo production

The aim of our study was to examine the influence of administration of N6,2’-dibutyryladenosine 3’5’-cyclic monophosphate (dbcAMP), a cAMP agonist, on ovarian folliculogenesis and atresia, as well as on reproductive efficiency in rabbits, whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by 20 IU/kg PMSG followed by 35 IU/kg hCG administration. Experimental animals received PMSG and hCG together with dbcAMP (at 5, 25 or 50 μg/animal). After ovulation and insemination, the animals were sacrificed. Ovaries were weighted, histological sections of ovaries were prepared, and the presence of ovulated and not ovulated follicles and different stages of atresia was evaluated by light microscopy. The eggs were flushed from the oviducts after insemination and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived zygotes and embryos reaching hatched blastocyst stage were determined. Administration of dbcAMP (at doses 25 or 50 μg/animal, but not at 5 μg/animal) was able to increase the proportion of follicles with cystic and luteinization-related atresia. Furthermore, dbcAMP (50 μg/animal, but not lower doses) increased the ovarian mass, number of Corpora lutea, number of harvested oocytes, zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that dbcAMP can stimulate rabbit ovarian follicle atresia, ovulation, oocyte, zygote and embryo yield and development. Furthermore, they confirm in the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of dbcAMP in improving animal reproduction and fertility

Uticaj transgenaze na kvalitet i prinos mesa kunića

In this paper results of the effect of transgenesis on quality and yield of rabbit meat are presented. During the trial body mass of transgenic progeny of F1 generation was monitored and compared to control group (nontransgenic animals of same age). Subsequent to slaughtering, meat yield, ratio between certain musculature parts and meat quality (proteins, lipids, water) were analyzed. Obtained data was compared to control group of animals of same age but standard genotype. Meat colour was evaluated on apparatus Specol 11 and expressed as percentage of remission on wave length of 540 μm. Content of elements in thigh muscle was established subsequent to dry mineralization in spectro-photometer UNICAM 939 Cambridge UK. Phosphorus content was measured spectro-photometrically on apparatus SPECOL 11. Subsequent to measuring and systematization, data was statistically analyzed and processed. Arithmetic mean values for certain groups of data were calculated, and their values compared using t-test (Hadživuković, 1991). Changes established in regard to content of water, lipids, energy and water binding capacity, were relative to changes in histological structure and level of metabolic processes. It is possible that these changes are result of pleiotropic effect of integrated gene. However, in order to confirm and interpret these changes, it is necessary to carry out further researches of the microscopic structure and metabolic processes of muscle tissues in transgenic rabbits.U radu su prikazani rezultati uticaja transgeneze na kvalitet i prinos mesa kunića. Ogled je vršen na komercijalnim tovnim hibridima nastalim ukrštanjem Novozelandskih belih i Kalifornijskih kunića. Dobijeni podaci upoređeni su sa kontrolnom grupom vršnjaka standardnog genotipa. Posmatrani su sledeći parametri kod obe grupe životinja: telesna masa (1, 2, 5, 10, 20 i 30- tog dana starosti), klanični podaci (w-težina pre žrtvovanja, dw- težina nakon iskrvarenja, s-težina kože, sp-težina distalnih delova zadnjih nogu, b- težina polutke sa kožom, hd-težina glave bez kože, fl- težina prednjih nogu, t- težina butova, r-težina rebara (grudi), bk - težina leđa, ht- težina srca, ky-težina bubrega, l- težina pluća, lvtežina jetre, git- težina stomaka i creva sa sadržajem, gite-težina praznog stomaka i creva, f- težina masnoće, bt- težina kostiju nogu, mt-težina mišića nogu, bf- težina kostiju prednjih nogu, c- prinos mesa (obraslost muskulaturom %); kvalitet mesa butova (cw- sadržaj vode, cp - sadržaj proteina, cf- sadržaj masti, ce- energija, pH, cc- boja, bw- kapacitet zadržavanja vode) i sadržaj mikroelemenata u mesu buta (Cu, Zn, Fe, K, Na, Mg, P, Ca). Posle merenja i sistematizacije podaci su statistički obrađeni. Izvršena su izračunavanja aritmetičkih sredina pojedinih grupa podataka, a zatim poređenje njihovih vrednosti t-testom (Hadživuković, 1991). Nizak nivo varijabiliteta u svim starosnim kategorijama u obe posmatrane grupe je jasno vidljiv iz vrednosti standardne greške aritmetičke sredine. To je manifestovano statistički značajnim uticajem procesa transgeneze na živu masu pri rođenju uprkos činjenici da je apsolutna razlika aritmetičkih sredina transgene i kontrolne grupe samo 0,005 kg (0,063±0,001 nasuprot 0,058±0,002) (tabela 1). Ovaj uticaj je na granici P=0,05 u tabeli analize varijanse (tabela 2). Razlika aritmetičkih sredina nije statistički značajna kod ostalih starosnih kategorija i ona se gubi već nakon 48 časova. Može se konstatovati da integrisani gen nema uticaja na porast transgenih kunića. Statistički značaj uticaja integrisanog gena je utvrđen kod parametara koji se odnose na masu distalnih delova transgenih i netransgenih kunića (0,062±0,001 nasuprot 0,069±0,001 kg), težine glave (0,119±0,003 nasuprot 0,128±0,003 kg) i težine butova (0,405±0,010 nasuprot 0,433±0,009 kg). Kod ostalih klaničnih karakteristika koje su testirane nisu utvrđene statistički značajne razlike koje bi bile uslovljene integracijom gena (tabele 3 i 4). Na uzorcima mesa nogu (butovi) praćene su vrednosti koje se odnose na kvalitet mesa (tabele 5 i 6). Dobijeni podaci koji se odnose na sastav mesa (tabela 5) ukazuju da je uticaj integracije gena bio statistički značajan (p (lt) 0,05) u grupi transgenih kunića u poređenju sa netransgenim u pogledu sledećih karakteristika: sadržaj proteina (74,03±0,26 nasuprot 74,84±0,28%), sadržaj masnoće (3,66±0,40 nasuprot 2,32±0,44%), sadržaja energije (495,43±11,81 nasuprot 458,07±12,94%), kapacitet zadržavanja vode (31,66±0,84 nasuprot 35,63±0,92%). Statistički značajne razlike kao posledica uticaja integrisanog gena nisu utvrđene kod ostalih posmatranih parametara (tabela 6). Srednje vrednosti sadržaja elemenata u tkivu mišića pokazale su najveće varijacije od svih posmatranih parametara. Najizraženije varijacije bile su u grupi netransgenih kunića (tabela 7). Broj životinja ili ponavljanja je igrao veliku ulogu. Za većinu posmatranih karakteristika može se reći da nisu pokazale uticaj integrisanog WAP- hFVIII gena u genotipu kunića. Značajnije razlike pojavile su se samo u okviru nekih parametara kvaliteta mesa (tabela 8). Rezultati ukazuju da nije utvrđeno ni prisustvo rhFVIII u skeletnim mišićima transgenih kunića

Effect of Nickel Administration in vivo on the Testicular Structure in Male Mice

The aim of this study was to describe the effects of nickel (NiCl2) on murine testicular structure. Experimental animals were injected intraperitoneally with a single dose of 20 mg NiCl2 per kg of body mass (group A, n = 5) and 40 mg NiCl2 per kg b. m. (group B, n = 5). The group without injection (n = 5) was the control (C). Animals were killed 48 hours after administration of nickel. The body mass of animals, the mass of testes and the testes : body mass ratio were not significantly affected. In both experimental groups a significant (p p p < 0.05 - 0.001) after nickel administration. Evaluation of the lumen diameter in the seminiferous tubule showed a significant increase in both experimental groups. The data of the perimeter of seminiferous tubules corresponded with those of the seminiferous tubule diameter. TUNEL assay detected a higher frequency of localized apoptosis in the interstitium of nickel-administered animals compared to control group. Our findings clearly suggest a negative effect of nickel on the structure as well as on the function of the seminferous epithelium at the site of spermatozoa production

THE EFFECT OF YUCCA SCHIDIGERA EXTRACT IN DIET OF RABBITS ON NUTRIENT DIGESTIBILITY AND QUALITATIVE PARAMETERS IN CAECUM

ABSTRACT The aim of this study was to determine the effects of yucca (Yucca schidigera) powder extract added to the rabbit feed mixtures. The animals of experimental group (EG1) were fed diet enriched with supplement of Yucca schidigera dry extract 5g/100 kg feed; rabbits of EG2 had diet enriched with yucca dry extract 20g/100 kg feed for 42 days. The control group (CG) was fed untreated pellet diet. The experiment was carried out on commercial hybrid Hycole rabbits. Between 65 and 70 days of age, 3 male rabbits from each group were selected for digestibility tests using the balance method. The faeces were collected individually during 5 consecutive days according to the European reference method for rabbit digestion trials. Chemical analysis of the diets and faeces for dry matter, crude protein, crude fibre, crude fat, nitrogen free extract, organic matter and starch was carried out according to the same method. The caecal samples from each of three slaughtered rabbits were collected for analysis; pH, VFA, ammonia-N and lactic acid were determined. The rabbits in our experiment showed the lowest coefficients of digestibility of crude fibre, and higher digestibility of crude protein, crude fat and organic matter, but the differences were not statistically significant comparing with control diet. The effect of yucca extract addition is manifested in reducing ammonia levels in the caecum of rabbits

Effects of dietary supplementation of nickel and nickel-zinc on femoral bone structure in rabbits

<p>Abstract</p> <p>Background</p> <p>Nickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits.</p> <p>Methods</p> <p>One month-old female rabbits were divided into three groups of five animals each. Group 1 rabbits were fed a granular feed mixture with addition of 35 g NiCl₂per 100 kg of mixture for 90 days. In group 2, animals were fed a mixture containing 35 g NiCl₂and 30 g ZnCl₂per 100 kg of mixture. Group 3 without administration of additional Ni or Zn served as control. After the 90-day experimental period, femoral length, femoral weight and histological structure of the femur were analyzed and compared.</p> <p>Results</p> <p>The results did not indicate a statistically significant difference in either femoral length or weight between the two experimental groups and the control group. Also, differences in qualitative histological characteristics of the femora among rabbits from the three groups were absent, except for a fewer number of secondary osteons found in the animals of groups 1 and 2. However, values for vascular canal parameters of primary osteons were significantly lower in group 1 than in the control one. Peroral administration of a combination of Ni and Zn (group 2) led to a significant decreased size of the secondary osteons.</p> <p>Conclusions</p> <p>The study indicates that dietary supplementation of Ni (35 g NiCl₂per 100 kg of feed mixture) and Ni-Zn combination (35 g NiCl₂and 30 g ZnCl₂per 100 kg of the mixture) affects the microstructure of compact bone tissue in young rabbits.</p

Differential interactions between IGFBP-3 and transforming growth factor-beta (TGF-β) in normal vs cancerous breast epithelial cells

In addition to modulating insulin-like growth factors action, it is now clear that insulin-like growth factor-binding protein-3 also has intrinsic effects on cell growth and survival. We have compared the effects of insulin-like growth factor-binding protein-3 and transforming growth factor-beta on cell proliferation and death of Hs578T cells and the normal breast epithelial cell line, MCF-10A. The growth of MCF-10A cells was inhibited at low concentrations of insulin-like growth factor-binding protein-3 but stimulated at high concentrations. These differential effects were unaffected in the presence of an insulin-like growth factor-I receptor antagonist. A synthetic peptide corresponding to the serine phosphorylation domain of insulin-like growth factor-binding protein-3 (that does not bind to insulin-like growth factors) also mimicked these differential actions. The growth of both cell lines was significantly inhibited by transforming growth factor-beta, this was associated with a 14-fold increase of insulin-like growth factor-binding protein-3 secreted by the Hs578T cells but a five-fold decrease of insulin-like growth factor-binding protein-3 secreted by MCF-10A cells. Replacement doses of exogenous insulin-like growth factor-binding protein-3 overcame the transforming growth factor-beta-induced growth inhibition in the MCF-10A cells. Cell death induced by ceramide was significantly reduced by insulin-like growth factor-binding protein-3 in the MCF-10A cells and depleting insulin-like growth factor-binding protein-3 with transforming growth factor-beta in these cells consequently increased their susceptibility to ceramide. In contrast, insulin-like growth factor-binding protein-3 enhanced apoptosis induced by ceramide in the Hs578T cells but transforming growth factor-beta treated Hs578T cells were resistant to apoptosis. The addition of anti-sense mRNA to insulin-like growth factor-binding protein-3 significantly abrogated this effect of transforming growth factor-beta. These data indicate that insulin-like growth factor-binding protein-3 has intrinsic activity capable of inhibiting or enhancing the growth and survival of breast epithelial cells depending on the cell line and exposure to other cytokines

Sex-related variation in compact bone microstructure of the femoral diaphysis in juvenile rabbits

<p>Abstract</p> <p>Background</p> <p>While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.</p> <p>Methods</p> <p>Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.</p> <p>Results</p> <p>Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.</p> <p>Conclusion</p> <p>The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.</p