The Isolation, Differentiation, and Survival In Vivo of Multipotent Cells from the Postnatal Rat filum terminale

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo

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

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

Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88017/1/j.1538-7836.2011.04505.x.pd

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]

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

State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

[EN] This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.This research was supported by the projects: UGAVIII/16/2015, VEGA 1/0611/15, by the Spanish Research project AGL2014-53405-C2-1-PComision Interministerial de Ciencia y Tecnologia (CICYT), Generalitat Valenciana research program (Prometeo II 2014/036), grant of Slovak Research and Development Agency: APVV-14-0043 and by the European Community under project no 26220220180: Building Research Centre "AgroBioTech". B. Kulikova received fellowship from a Collaborative European Network on Rabbit Genome Biology (RGB-Net) (COST-STSM-TD1101)Kulikova, B.; Jiménez-Trigos, ME.; Makarevich, AV.; Chrenek, P.; Vicente Antón, JS.; Marco-Jiménez, F. (2016). State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes. Cryobiology. 72(1):14-20. https://doi.org/10.1016/j.cryobiol.2015.11.009142072