Distinct dynamics of lipid accumulation by porcine cumulus cells during in vitro maturation with follicular fluid of low and high fatty acid contents.

Follicular fluid (FF) is a complex biological medium providing a fully balanced microenvironment for the oocyte. The standard medium for in vitro maturation of porcine oocytes contains 10% of FF which due to its unknown and inconstant composition is a source of a significant variation. This study aimed to investigate whether follicular fluids of significantly different fatty acid contents (standardized follicular fluid) supplemented to porcine IVM media affects lipid metabolism of porcine oocytes and cumulus cells. Two categories of FF from cyclic gilts containing high (H) and low (L) FA content was added to IVM medium for oocytes from prepubertal gilts. Altogether, 521 cumulus oocyte-complexes (oocytes and corresponding cumulus cells) were analyzed for mRNA expression of 7 genes regulating lipid metabolism and selected traits of the lipid droplets (LD). The applied FFs of different FA levels exerted distinct effects on oocytes and cumulus cells (CCs). During IVM oocytes tended to utilize the lipids as demonstrated by the reduced LD number and lipid fluorescence, whereas cumulus cells accumulated lipids as indicated by the increase in LD number, the occupied area and fluorescence level. Changes in cumulus cells were independent of the FA content in the follicular fluid which means an efficient lipid accumulation during IVM. Final analysis including the effect of FA level on LD traits in oocytes and corresponding CCs revealed two distinct patterns. COCs matured in FF of high FA content were characterized by elevated dynamics of lipid accumulation in CCs and stable lipid content in oocytes. In the case of FF with low FA content, CCs accumulated lipids at a significantly lower rate whereas lipid level in oocytes was reduced. The alterations observed in the LD parameters were not accompanied by changes in oocyte nuclear maturation and in transcript level of any from the 7 analyzed genes. In conclusion, fatty acid content of the follicular fluid supplemented to porcine IVM medium affects lipid metabolism in cumulus cells of the maturing oocyte and application of a standardized FF may help to improve the quality of porcine oocytes matured in vitro.

Prepubertal heifers versus cows-The differences in the follicular environment.

The oocyte quality is to a large extent influenced by the sexual maturity of the donor female. Although this phenomenon has already been broadly described in domestic animals, the underlying mechanisms are poorly understood. Published data focus on oocyte ultrastructure, fertilization abnormalities, and blastocyst developmental rate. The goal of the present experiment was to characterize the follicular environment (oocyte, cumulus [CC] and granulosa (GC) cells as well as follicular fluid [FF]) in ovarian follicles of prepubertal heifers and cows. Each experimental replicate included the following set of traits within individual follicles: lipid droplets (LDs) number in oocytes, expression of seven genes involved in energy metabolism (fatty acids [FAs] metabolism-ELOVL2, ELOVL5, SCD, FADS2, glucose transport-GLUT1, GLUT3, GLUT8) in CC and GC as well as FA composition and glucose concentration in FF. According to our results, cow oocytes were larger in diameter and contained more LD than those from prepubertal heifers, both before and after IVM. The LD number was also higher in cow oocytes after IVM, when compared to immature oocytes. The FF from cow follicles had elevated glucose content similarly to the majority of the analyzed FA. Transcript analysis revealed differences for five out of seven analyzed genes (ELOVL, FADS2, SCD, GLUT3, GLUT8) in CC and GC cells. However after considering the female category, the only difference was noticed for the mRNA of SCD gene, which was more abundant in cow GC. This finding may indicate distinct roles of CC and GC in follicular energy metabolism. In conclusions, we suggest that distinct properties of follicular environment in prepubertal heifers and cows may be responsible for differences in the quality of oocytes from the two categories of donors. We hypothesize that suboptimal environment in heifer follicles (glucose and FA lower content in FF) determines reduced quality of their oocytes (lower diameter and LD number) and limited maturation potential. Besides, energy demands of heifer oocytes may be restricted due to a low LD number, exerting a negative effect on the development of the future embryo. The advantages of cow gametes (e.g., higher LD number and diameter) attributed to oocytes of superior quality may support the statement that cows donate oocytes of better quality than heifers.

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.

Transcript abundance, glutathione and apoptosis levels differ between porcine oocytes collected from prepubertal and cyclic gilts.

It is well known that puberty has a strong impact on oocyte developmental competence in vitro; however, the reason for this phenomenon at the cellular level has not been clarified yet. It is hypothesized that cytoplasmic maturation is responsible for oocyte quality and may be impaired in prepubertal gilts. Previous results on mitochondrial DNA copy number and mitochondria and cortical granule distribution showed that cytoplasmic maturation is a complex trait and should include multithreaded analysis. Therefore, the aim of the present research was to analyze the transcript abundance of developmentally important genes (BMP15, GDF9, GSTA2, ATP5A1, EEF1A1, BAX, BCL2) followed by investigation of the glutathione and apoptosis level in oocytes of prepubertal and cyclic gilts. We found differences in relative transcript abundance of BMP15 and GDF9 genes after IVM, whereas different concentrations of glutathione were noted before IVM (5.3 vs. 2.9 pmol, respectively). The glutathione level was equalized after IVM (10.3 vs. 9.1 pmol), whereas the incidence of apoptosis remained similar before (3.9% vs. 1.1%) and after IVM (4.5% vs. 1.9%) being higher in prepubertal oocytes. A potential impact of gilt puberty on oocyte quality has been therefore masked by the significant effect of IVM. Because the maternal effect genes, BMP15 and GDF9, play key roles in regulation of folliculogenesis and oocyte-cumulus interaction, their upregulation in oocytes of cyclic gilts may result in increased developmental competence. On the basis of findings from this and our previous research, we suggest that the reduced quality of oocytes from prepubertal females is a complex phenomenon and is not related to a single marker trait.

Short-term storage and swim-up selection do not affect the x/y ratio in equine spermatozoa.

The standard procedure of artificial insemination with fresh equine spermatozoa involves short-term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short-term storage and the swim-up procedure. We used a standard protocol, for short-term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro™ (Minitüb GmbH, Tiefenbach, Germany). After each set-up storage period, the motile fraction of sperm cells was selected by the swim-up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non-selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim-up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species-related differences.

Three-dimensional positioning of B chromosomes in fibroblast nuclei of the red fox and the chinese raccoon dog.

Great progress has been achieved over the last years in studies on chromosome arrangement in mammalian cell nuclei. Growing evidence indicates that the genome's spatial organization is of functional relevance. So far, no attention has been paid to the nuclear organization of B chromosomes (Bs). In this study we have examined nuclear positioning of Bs in 2 species from the Canidae family--the red fox and the Chinese raccoon dog. Using 2D and 3D fluorescence in situ hybridization and 2 gene-specific probes (C-KIT and PDGFRA), we analyzed the location of Bs in fibroblast nuclei. We found that small Bs of the red fox occupied mostly the interior of the nucleus, while medium-sized Bs of the Chinese raccoon dog were observed in the peripheral area of the nucleus as well as in intermediate and interior locations. The more uniform distribution of B chromosomes in the Chinese raccoon dog may be the result of differences in their size, since 3 morphological types of Bs are distinguished in this species. Our results indicate that 3D positioning of B chromosomes in fibroblast nuclei of the 2 canid species is in agreement with the chromosome size-dependent theory.

No single way to explain cytoplasmic maturation of oocytes from prepubertal and cyclic gilts.

The objective of this study was to evaluate selected aspects of cytoplasmic maturation in oocytes from prepubertal and cyclic crossbred gilts before and after in vitro maturation. For this purpose, cortical granule redistribution, mitochondrial DNA content and mitochondria translocation were analyzed. Moreover, for the first time the fatty acid profiles in follicular fluid (FF) of both gilt categories was evaluated. The nuclear maturation (the percentage of metaphase II oocytes was 83% in prepubertal gilts compared with 87% in cyclic gilts), cortical granule relocation from the cortex to peripheral ooplasm (98.7% vs. 98.8% of oocytes, respectively) and mitochondrial DNA content (227 543 vs. 206 660, respectively) was not affected by sexual maturity of the donor gilt. However, the redistribution of active mitochondria during in vitro maturation was observed only in the oocytes of cyclic gilts. With regard to FF analysis, saturated, unsaturated, and monounsaturated fatty acids were significantly more abundant in the FF of prepubertal females. In particular, stearic (C18:0) and palmitic (C16:0) fatty acids had significantly higher concentrations in the FF of prepubertal gilts. In conclusion, although the oocytes of prepubertal gilts matured in vitro at a rate similar to those of cyclic gilts, they differed with respect to the selected factors attributed to cytoplasmic maturation. We suggest that the higher content of particular fatty acids, which is known to have a negative influence on oocyte maturation, as well as impaired mitochondria redistribution are factors limiting the maturation potential of oocytes from prepubertal gilts.

Early cleaved bovine embryos show reduced incidence of chromosomal aberrations and higher developmental potential on day 4.5 post-insemination.

The present study aimed to investigate whether the timing of the first zygotic cleavage (FZC) influences the speed of embryo development expressed by the total cell count and the rate of chromosomally aberrant embryos. Bovine embryos were produced in vitro and divided into two categories according to the timing of FZC: early cleavers (at 30 hpi; EC) and non-early cleavers (at 48 hpi; NEC). On day 4.5 pi, embryos were grouped into three classes depending on the number of blastomeres: delayed (<8 BL), normal (8-16 BL) and advanced (>16 BL). We applied fluorescence in situ hybridization (FISH) with probes for bovine chromosomes 6 and X. The only form of chromosomal imbalance observed was mixoploidy [(2n/3n; 2n/4n); 19.9%, 54/271]. Early cleavers were less often chromosomally unbalanced (13.9%, 20/144) than their NEC counterparts (26.7%, 34/127). Among embryos developing at a normal speed, the NEC embryos were more often abnormal (NEC 20/80; EC 10/79; p < 0.05). The advanced embryos were not observed among the NEC category, whereas such embryos from EC category displayed no chromosomal aberrations. The majority of embryos arrested at the 8 BL stage were of NEC category and were carriers of chromosomally abnormal blastomeres. With regard to embryonic sex, we demonstrated that although males dominate among bovine embryos developing in vitro, the incidence of mixoploidy was equal for both sexes. It can be suggested that a good-quality bovine embryo is usually an early cleaver that develops at higher speed and contains less aberrant cells. The present study also confirmed the usefulness of the FZC as a marker of embryo quality by demonstrating a significantly lower incidence of aberrations in early embryos.

Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts.

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.