The impact of retrieval method and breed on the motility and kinematic parameters of fresh and post-thaw ram epididymal spermatozoa.

This study was conducted to develop ideal post-mortem gamete retrieval and conservation methods to establish a Hungarian ex-situ in vitro gene bank. Pairs of testes from German Mutton Merino (n = 7) and Hungarian Black Racka (n = 7) rams were collected at a slaughterhouse, transported to the laboratory and stored overnight (4-5 °C) before processing. Post mortem ram epididymal spermatozoa (REPS) were obtained from the cauda epididymidis by slice or incision methods. Fresh samples were extended to 200 × 106/mL cell concentration, filled into mini straws and equilibrated at 5 °C for 2 h. Freezing was performed manually in a Styrofoam box. The fresh and post-thaw total motility, progressive motility and kinematic parameters of REPS were assessed using the CASA technique. The collection method did not affect significantly the fresh and post-thaw motility and kinematic parameters. Merino had higher (P < 0.05) testicular weight. Racka had significantly better fresh and post-thaw linear movement but had statistically the same (P > 0.05) cryotolerance as Merino. In conclusion, both collection methods were found suitable for REPS retrieval. The REPS from Racka exhibited better linear movement values than those from the Merino breed. The cryotolerance of REPS of both breeds was comparable.

Effect of single-nucleotide polymorphisms on specific reproduction parameters in Hungarian Large White sows.

The aim of this study was to reveal the effect of single-nucleotide polymorphisms (SNPs) on the total number of piglets born (TNB), the litter weight born alive (LWA), the number of piglets born dead (NBD), the average litter weight on the 21st day (M21D) and the interval between litters (IBL). Genotypes were determined on a high-density Illumina Porcine SNP 60K BeadChip. Data screening and data identification were performed by a multi-locus mixed-model. Statistical analyses were carried out to find associations between individual genotypes of 290 Hungarian Large White sows and the investigated reproduction parameters. According to the analysis outcome, three SNPs were identified to be associated with TNB. These loci are located on chromosomes 1, 6 and 13 (-log10P = 6.0, 7.86 and 6.22, the frequencies of their minor alleles, MAF, were 0.298, 0.299 and 0.364, respectively). Two loci showed considerable association (-log10P = 10.35 and 10.46) with LWA on chromosomes 5 and X, the MAF were 0.425 and 0.446, respectively. Seven loci were found to be associated with NBD. These loci are located on chromosomes 5, 6, 13, 14, 15, 16 and 18 (-log10P = 10.95, 5.43, 8.29, 6.72, 6.81, 5.90, and 5.15, respectively). One locus showed association (-log10P = 5.62) with M21D on chromosome 1 (the MAF was 0.461). Another locus was found to be associated with IBL on chromosome 8 (-log10P = 7.56; the MAF was 0.438). The above-mentioned loci provide a straightforward possibility to assist selection by molecular tools and, consequently, to improve the competitiveness of the Hungarian Large White (HLW) breed.

X- and Y-chromosome-specific variants of the amelogenin gene allow non-invasive sex diagnosis for the detection of pseudohermaphrodite goats.

The Polled Intersex Syndrome (PIS) is responsible for the absence of horns in homozygous and heterozygous goats causing a female-to-male sex reversal in the homozygous polled genotypic female (XX) goats. A simple and efficient non-invasive method was elaborated to detect the genotypic sex from hair and faecal samples using a pair of primers to amplify the X- and Y-linked alleles of the amelogenin gene. The PCR products were easily distinguishable using agarose gel electrophoresis: we detected an X-specific single band in samples originating from healthy phenotypic females and double (X- and Y-) bands in samples from males. The new PCR method is applicable for diagnosing the sex of PIS-affected animals already as newborn kids, in contrast with the phenotypic findings appearing only after puberty, and thus it may replace the cumbersome chromosome investigations.

Leukemia inhibitory factor promotes porcine oocyte maturation and is accompanied by activation of signal transducer and activator of transcription 3.

We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22 hr, last 22 hr, or entire 44 hr duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P < 0.05) and obvious induction of cumulus expansion were observed over the whole time span (44 hr) in the IVM group. When cumulus cells were removed at 22 hr and denuded oocytes were further cultured, pLIF showed no effect on maturation rate. Oocytes matured in pLIF-supplemented medium showed a tendency for more rapid blastocyst development (21.1% vs. 16.2%, P = 0.0715). Examination of transcripts and proteins of the LIF signaling pathway in COCs revealed that LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) are present in both cumulus cells and oocytes. The amount of phosphorylated STAT3 (p-STAT3) markedly increased in both cumulus cells and oocytes cultured in pLIF-supplemented media, although oocyte p-STAT3 disappeared after 44 hr of IVM. These results suggest that the LIF/STAT3 pathway is functional during IVM of porcine oocytes, and supplementing pLIF in the IVM medium can improve oocyte maturation by activating this pathway.

The Influence of Three Commercial Soy Lecithin-Based Semen Extenders and Two Spermatozoa Concentrations on the Quality of Pre-Freeze and Post-Thaw Ram Epididymal Spermatozoa.

There are limited studies on the factors affecting the success of ram epididymal spermatozoa (REPS) cryopreservation. On this note, the current study assessed the influence of three commercial soy lecithin-based semen extenders, AndroMed® (AND), BioXcell® (BIO), and OviXcell® (OVI), and two concentrations (400 × 106 vs. 200 × 106 spermatozoa/mL) on the pre-freeze and post-thaw quality of REPS. The REPS were retrieved from nine adult rams' testes and diluted with each of the three extenders to both concentrations. Straws were frozen manually. Standard motility (SMP) and kinematic parameters (KPs) were assessed via a CASA, while spermatozoa viability, morphology, and acrosomal integrity were assessed via the Kovács-Foote staining technique. The concentration did not significantly affect the pre-freeze and post-thaw SMP and KPs of REPS. BIO and OVI had significantly higher pre-freeze and post-thaw BCFs, post-thaw VAP, and the percentage of all intact heads than AND. In contrast, AND had a significantly lower percentage of REPS with tail defects than BIO and OVI. The 400 × 106 spermatozoa/mL concentration resulted in a significantly higher percentage of all intact heads than the 200 × 106 spermatozoa/mL concentration. Freezing significantly increased tail defects and decreased the percentage of REPS with distal cytoplasmic droplets. The cryopreservation of REPS at the 400 × 106 spermatozoa/mL concentration is recommended. All three extenders must be optimized to preserve the viability, membrane integrity, and better normal morphology of REPS; the reason for increased tail abnormality after the freezing/thawing process needs to be studied.

Noninfectious Causes of Pregnancy Loss at the Late Embryonic/Early Fetal Stage in Dairy Cattle.

In cattle, initial pregnancy diagnosis takes place during the late embryonic/early fetal stage of gestation. From this point onward, pregnancy loss may occur in up to one fifth of pregnancies before the initial pregnancy diagnosis is confirmed. This means the early identification of risk factors is a key part of pregnancy diagnosis and herd management. The various factors responsible for pregnancy losses are classified into infectious and noninfectious. Among the noninfectious causes, several dam-related (circumstances of the individual pregnancy or milk production) and herd-related factors causing stress have been well established. In this review, we summarize the impacts of these noninfectious factors and predict associated risks of pregnancy loss.

Salivary cortisol as a non-invasive approach to assess stress in dystocic dairy calves.

The intensity and the magnitude of saliva cortisol responses were investigated during the first 48 h following birth in newborn dairy calves which underwent normal (eutocic, EUT, n = 88) and difficult (dystocic, DYS, n = 70) calvings. The effects of parity and body condition of the dam, the duration of parturition, the time spent licking the calf, the sex and birth weight of the calf were also analyzed. Neonatal salivary cortisol concentrations were influenced neither by factors related to the dam (parity, body condition) nor the calf (sex, birth weight). The duration of parturition and the time spent licking the calf also had no effect on salivary cortisol levels. Salivary cortisol concentrations increased rapidly after delivery in both groups to reach their peak levels at 45 and 60 min after delivery in EUT and DYS calves, respectively supporting that the birth process means considerable stress for calves and the immediate postnatal period also appears to be stressful for newborn calves. DYS calves exhibited higher salivary cortisol concentrations compared to EUT ones for 0 (P = 0.022), 15 (P = 0.016), 30 (P = 0.007), 45 (P = 0.003), 60 (P = 0.001) and 120 min (P = 0.001), and for 24 h (P = 0.040), respectively. Peak levels of salivary cortisol and the cortisol release into saliva calculated as AUC were higher in DYS than in EUT calves for the 48-h of the sampling period (P = 0.009 and P = 0.003, respectively). The greater magnitude of saliva cortisol levels in DYS calves compared to EUT ones suggest that difficult parturition means severe stress for bovine neonates and salivary cortisol could be an opportunity for non-invasive assessment of stress during the early neonatal period in cattle.

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro.

BACKGROUND: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. RESULTS: Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. CONCLUSION: Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.

Production of identical mouse twins and a triplet with predicted gender.

The aim of this study was to develop a method to generate identical twins and triplets with predicted gender. As a first step toward that aim, single blastomeres obtained from EGFP expressing eight-cell stage embryos and either diploid or tetraploid host embryos were used to compose chimera. We could follow the fate of EGFP expressing diploid blastomere derived cells in 3.5- and 4.5-day-old chimera embryos in vitro. We found that the diploid blastomere-derived cells had significantly higher chance to contribute to the inner cell mass if tetraploid host embryos were applied. After that, we developed a quick and reliable multiplex PCR strategy for sex diagnosis from single blastomeres by simultaneous amplification of the homologous ZFX and ZFY genes. By composed chimeras using single blastomeres, derived from sexed eight-cell stage embryos and a tetraploid host embryo, we could get preplanned sex newborns, wholly derived from these blastomeres. Among these mice, identical twins and a triplet were identified by microsatellite analysis. Unlike clones produced by nuclear transfer, these mice are identical at both the nuclear as well as mitochondrial DNA level. Therefore, the tetraploid embryo complementation method to produce monozygotic twins and triplets could be a valuable tool both in biomedical and agricultural applications.

Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits.

The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species. Here we clearly show the differential expression of Venus reporter protein during spermatogenesis of the two compared species, the laboratory rabbit and mice. We provide evidence for the functionality of intercellular bridges in the male germline and genotype-independent transgenic phenotype of rabbit spermatids. Our data suggest that the reporter rabbit line may be a suitable tool to identify molecular mechanisms in testicular development, and may contribute to develop better animal models for male infertility in men.

Experiences in deer sperm cryopreservation under practical conditions--a pilot study.

The genetic potential of the red and fallow deer populations in Hungary is well known. Conserving the variability in this excellent genetic material for game preservation is one of our most important task. The aim of the present pilot study was to test the logistical steps of a sperm processing and storing system in which deer sperm can be stored at a level that meets quality standards accepted for domestic animals. Moreover, two different semen extenders, commercially used for freezing bull semen, were compared from the viewpoint of applicability to freeze fallow deer sperm. Sperm was collected from epididymes of eight red stags (Cervus elaphus hippelaphus) and six fallow bucks (Dama dama) during the rutting season. Red deer samples were washed in Triladyl extender, while fallow deer samples were split and processed in Triladyl or Bioxcell extender. In the samples, which had a shorter time interval between the death of the animal and the sperm collection, the percentage of viable spermatozoa with intact acrosome was typically higher.

Generation of mouse embryonic stem cell lines from zona-free nuclear transfer embryos.

Pluripotent stem cells would have great potential in cell therapies and drug development when genetically matched with the patient; thus, histocompatible cells could be used in transplantation therapy or as a source of patient-specific cells for drug testing. Pluripotent embryonic stem cells (ESCs)-generated via somatic cell nuclear transfer (SCNT) or parthenogenesis (pESC)-are potential sources of histocompatible cells and tissues for transplantation. Earlier studies used the piezoelectric microinjection (PEM) technique for nuclear transfer (NT) in mouse. No specific studies examined zona-free (ZF) NT as an alternative NT method to generate genetically matched ESCs of a nuclear donor. In this study, we compared the efficiency of nuclear transfer-derived ESC (ntESC) line establishment from ZF-NT, ZF-parthenogenetic (PGA), and ZF-fertilized embryos with that of the PEM-NT method. Different nuclei donor cells [cumulus, ESC, and mouse embryonic fibroblast (MEF)] were used and the efficiency of ntESC derivation was investigated, along with their in vitro characterization. The ZF-NT method's efficiency was higher than that of the PEM-NT using cumulus cells. When ESCs and cumulus cells were used as nuclear donor cells, they resulted in significantly higher ZF-NT-derived ntESC line establishment rates compared to MEF cells. In conclusion, the nuclear donor cell type significantly affected the efficiency of ntESC line establishment, and the ZF-NT method was efficient to establish pluripotent ntESC lines.

Cotransfer of parthenogenetic embryos improves the pregnancy and implantation of nuclear transfer embryos in mouse.

The majority of somatic cell nuclear transfer (SCNT) clones dies in the peri- or postimplantation period. Improvement of the full-term healthy pregnancy rates is a key issue for the economical viability and animal welfare profile of SCNT technology. In this study the effects of cotransfer of parthenogenetic or fertilized embryos on the pregnancy and implantation of SCNT mouse embryos have been investigated. SCNT embryos were produced by transferring cumulus cell nuclei into enucleated B6D2F1 mouse oocytes, whereas parthenogenetically activated (PA) and fertilized embryos were derived from ICR mice by artificial activation with strontium and in vivo fertilization, respectively. SCNT embryos were inferior in their developmental capacity to blastocyst compared to either PA or fertilized embryos. SCNT embryos were transferred alone (SCNT), or cotransferred with two to three PA (SCNT + PA) or fertilized (SCNT + Fert) embryos into the oviducts of an ICR recipient. Both pregnancy and implantation rates originating from clones in the SCNT + PA group were significantly higher than those of SCNT group (p < 0.05). The weight of placentas of clones derived from SCNT, SCNT + PA, or SCNT + Fert was in all cases significantly higher than that of fertilized controls (p < 0.001). Most of the clones derived from SCNT embryos cotransferred with PA or fertilized embryos survived to adulthood and were fertile and healthy according to histopathological observations. Our results demonstrate in mouse that cotransfer of PA embryos improves the pregnancy and implantation of SCNT embryos without compromising the overall health of the resulting clones.

Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos.

The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (beta-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of beta-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on beta-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage.

Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays.

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to compare the gene expression patterns, and identify those most affected. For this, 8-cell stage embryos were collected from ICR mice and vitrified with solid surface vitrification technique, while maintaining equal numbers of embryos as control. Total RNAs were extracted and two rounds of amplification were employed. Finally three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22 K oligonucleotide slides and the results were analyzed with subsequent verification by independent real-time PCR analyses. The two rounds of amplification with 5 ng tRNA input have yielded 15-16 microg of cRNA. The analyses of repeated hybridizations showed 20,183 genes/ESTs as common signatures, and unsupervised analysis identified 628 differentially expressed (P < 0.01) genes. However, with at least 1.5-fold change considerations, 183 genes were differentially expressed (P < 0.01) out of which 107 were upregulated. The independent analysis with real-time PCR and unamplified samples fully confirmed the results of microarray, indicating the linearity of amplification. Furthermore, this novel gene expression study for vitrified embryos identified many new candidate genes with overrepresentation in some important biological processes. Thus, it is possible to conclude that the expression pattern reflected a broad spectrum of consequences of vitrification on embryos, with most effects on metabolism, regulatory role and stress response genes and allowed the identification of new candidate marker genes for cryosurvival.

Production of transgenic chimeric rabbits and transmission of the transgene through the germline.

Here we report that improved reproductive technologies combined with an efficient microinjection method and in vitro cultivation medium enabled us to create germ line chimeric rabbits. To follow the fate of the chimeric embryo a blastomere marked with the human blood coagulation factor VIII (hFVIII) transgene was microinjected into a morula stage wild type embryo. The degree of chimerism in different tissues was estimated by real-time PCR and was found to be in the range of 0.1-42%. Among the four chimeric animals, one was identified as a chromosomal intersex and two were germline chimeras.