István Egerszegi

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T(0)), after 22 h of in vitro maturation (IVM) (T(22)) and after 44 h of IVM (T(44)) (n = 496). BCB-stained oocytes (BCB+) at T(0) were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB-) oocytes at T(0) contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB- group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB- oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Abstract Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.

Reproductive function of Hungarian Mangalica boars: Effect of seasons

Seasonal changes in testis volume, testosterone (T) productivity (GnRH test) and semen characteristics of Mangalica boars were studied. The biggest testis volume was measured in autumn and the smallest in winter. Significant differences were demonstrated between autumn-winter (P = 0.012) and autumn-spring (P = 0.015) in testis volume. The highest basic T concentration (Tb) was observed in autumn and the lowest in summer. The provoked T concentration (Tincr) was significantly higher in autumn than in spring (P = 0.0007). A strong correlation was observed between T concentrations and testis volume in spring. The highest ejaculate volume was measured in winter while the lowest in autumn. Significant differences were found in semen concentration as well as in the total number of spermatozoa per ejaculate between seasons. The highest number of abnormal sperm cells was observed in spring while the lowest in summer. It can be concluded that the ejaculate of the Mangalica breed tends to be of lower volume and higher sperm concentration as compared to most pig breeds. Seasonal differences could be observed in testicular measurements, testosterone production capacity and sperm morphological features; however, sperm motility remained constantly high during the study.

Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period

The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5×106 cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by Čeřovský staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P<0.05) but not between the oviductal sections of each group (P>0.05). Compared with the MID group, the percent of intact sperm cells was higher (P<0.01) in the PERI and POST groups (32.8 vs. 66.4 and 76.8%). Also, the percentages of aberrations in the acrosome and tail were higher (P<0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.

Saving genetic resources of native pigs in occidental and oriental countries - Practical examples of the characterization and utilization of native pigs in Hungary and Laos

Worldwide, only a few “fatty” pig breeds exist with different and/or regional utilization. Using the Hungarian Mangalica, which almost went extinct in Europe and the Lao Moo Lat pig, which still has a large population in South-East Asia as exemples, we wanted to demonstrate that indigenous (fatty) pig breeds may represent both national value and tremendous economic potential. Since these less prolific and less productive breeds cannot contribute to mass production, new market roles and methods should be established for them in the premium segment of pork trading. Thus their preservation and propagation needs the comprehensive collaboration of commercial, governmental actors and researchers. Briefly summarizing the history, we report the current results of reproductive physiology research. The commercial renaissance of Mangalica pigs is indebted to the enthusiastic efforts of basic scientists, pig breeding experts and dedicated Mangalica producers. Scientific achievements were applied to practical breeding and production of delicious pork and processed products, which ultimately made the economic success in the Mangalica sector possible. Both, research on and utilization of endangered (pig) breeds maintain not only breed diversities, but also may improve the livelihood of farmers worldwide.

Looking for breed differentiating SNP loci and for a SNP set for parentage testing in Mangalica

Abstract. The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the possibility (1) to replace the previously applied ten microsatellite markers by nine SNP loci to classify the Blond, Swallow-Belly and Red Mangalica individuals into three different breed groups (P>0.95); (2) to propose 54 SNP loci for parentage testing in Mangalica pigs where the exclusion probability is 0.999115 if one parent is known and the probability of identity is 1.54×10-23.

Detection of pregnancy in sheep using an ELISA for pregnancy-specific protein B

The early detection of pregnancy and the determination of fetal numbers have economic benefits in sheep production because of the seasonal breeding patterns where missing a breeding opportunity means the loss of one productive year. The purpose of this study was to evaluate the efficacy of the B6-HRP ELISA for ovine pregnancy-specific protein B (oPSPB) measurement in the detection of pregnancy and estimation of fetal numbers in different sheep breeds. BioPRYN® ELISA assay kit was used for the detection of pregnancy in the experimental animals. Ninety-three ewes of three breeds (British Milksheep - BM, Lacaune - L and Transylvanian Racka - TR), each from three farms in Hungary, were included in the study. BM and L ewes were artificially inseminated (AI). Thirty-five days after AI, all ewes were examined by transabdominal ultrasound. The TR flock was mated naturally over a six-week period. At the end of the mating period, the ewes were similarly examined by ultrasound. Blood samples were taken from all pregnant ewes twice (35 and 65 days after AI), and serum samples were assayed by the BioPRYN test. It can be concluded that the detection of serum PSPB by ELISA is a much easier, safer, less expensive and highly accurate method for the detection of ovine pregnancy. Although some breed-related differences were detectable at 35 and 65 days post breeding, no differences in oPSPB levels were found in pregnant ewes carrying different numbers of fetuses.

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.