A comparison of the biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state at 5 °C.

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage.

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm.

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

Carbon Monoxide (CO) as a Retinal Regulator of Heme Oxygenases -1, and -2 (HO's) Expression.

Carbon monoxide (CO) has been proposed as a chemical light signal and neural system modulator via heme oxygenases -1 and -2 (HO-1 and HO-2). Many papers have proven the CO-HO circuit to be important for such physiological pathways as the molecular biological clock and the GnRH axis, but also in such pathological occurrences as ischemic injuries, or inflammation as a regenerative and neuroprotective factor. In this in vivo experiment, we used three groups of pigs: control-housed in natural conditions without any procedures; without CO-adapted and kept in constant darkness, infused with blank plasma; and with CO-adapted and kept in constant darkness infused with CO-enriched plasma. After the experiments, each animal was slaughtered and its eyes were collected for further analysis. Quantitative PCR and Western blot analysis were performed to show statistical differences in the expressions between the experimental groups. Our data revealed that exogenous CO is regulator of mRNA transcription for HO-1 and HO-2 and PCNA. Moreover, the mRNA abundance of analyzed factors in the experimental group after CO elevation revealed a restored gene-expression level similar to the control group, which we had observed in the group's restored protein level after CO elevation. In conclusion, exogenous CO regulates HO's and PCNA gene expression on transcriptional and translational levels in a similar way as a light cue.

Role of Methylation in Period2 (PER2) Transcription in the Context of the Presence or Absence of Light Signals: Natural and Chemical-Studies on the Pig Model.

It has been proposed that carbon monoxide (CO) is a chemical light carrier that is transferred by the humoral pathway from the retina to the brain. Here, we aimed to study how deeply CO is involved in regulating the expression of Period2 gene (PER2), one of the genes maintaining the intrinsic biological clock. In our in vivo experiment, we studied whether CO may be a chemical signal and is also equivalent to natural light in three groups of pigs: Normal: housed in natural conditions without any procedures, Control: adapted and kept in constant darkness, infused with blank plasma, and CO treated: adapted and kept in constant darkness infused with CO-enriched plasma. After the experiment, the animals were slaughtered at two times of day: 12 p.m. and 12 a.m. Next, hypothalamus samples were collected. Quantitative PCR, the DNA methylation of the promoter sequence containing enhancers (E-box) and a functional analysis of the PER2 promoter was performed. qPCR showed a differential pattern of PER2 mRNA expression at daytime oscillation in the examined groups. Pyrosequencing revealed daytime changes in the methylation level of regulatory sites of the examined sequence. Luciferase reporter assay confirmed that E-boxes (CANNTG) drive the expression of the porcine PER2 in vitro. In conclusion, changes in methylation over 24 h may regulate the oscillatory manner of PER2 expression.

Regulatory Potential of Long Non-Coding RNAs (lncRNAs) in Boar Spermatozoa with Good and Poor Freezability.

Long non-coding RNAs (lncRNAs) are suggested to play an important role in the sperm biological processes. We performed de novo transcriptome assembly to characterize lncRNAs in spermatozoa, and to investigate the role of the potential target genes of the differentially expressed lncRNAs (DElncRNAs) in sperm freezability. We detected approximately 4007 DElncRNAs, which were differentially expressed in spermatozoa from boars classified as having good and poor semen freezability (GSF and PSF, respectively). Most of the DElncRNAs were upregulated in boars of the PSF group and appeared to significantly affect the sperm's response to the cryopreservation conditions. Furthermore, we predicted that the potential target genes were regulated by DElncRNAs in cis or trans. It was found that DElncRNAs of both freezability groups had potential cis- and trans-regulatory effects on different protein-coding genes, such as COX7A2L, TXNDC8 and SOX-7. Gene Ontology (GO) enrichment revealed that the DElncRNA target genes are associated with numerous biological processes, including signal transduction, response to stress, cell death (apoptosis), motility and embryo development. Significant differences in the de novo assembled transcriptome expression profiles of the DElncRNAs between the freezability groups were confirmed by quantitative real-time PCR analysis. This study reveals the potential effects of protein-coding genes of DElncRNAs on sperm functions, which could contribute to further research on their relevance in semen freezability.

Determination of the activity and relative abundance of mRNA for antioxidant enzymes in stallion testicular and epididymal tissues: A comparison between two breeding seasons.

The key prerequisite for successful insemination is sperm characterized to have positive values for morphological and biological variables which are determined by, among others, effective antioxidant protection during the lifespan of sperm cells. This study evaluated the activity and relative abundance of mRNA for antioxidant enzymes in stallion testicular and epididymal tissues during breeding (n = 5) and non-breeding (n = 5) seasons. The activity of superoxide dismutase (SOD) was greater (P < 0.05) during the breeding season, in particular in the testes and the caput epididymis, and SOD1 was the predominant isoform of the enzyme. The expression of the SOD3 gene was markedly less in the analyzed tissues, which indicates that this enzyme contributes to the antioxidant protection of the stallion reproductive tract. The activity of catalase (CAT) was less (P < 0.05) in the testes during both seasons while its relative abundances only during the breeding season. The greatest CAT activity was noted in the cauda epididymis during the breeding season. The activity of glutathione peroxidases (GPx) was greater (P < 0.05) in the testes than in other tissues and 10-fold greater during the breeding season. Similarly, relative abundance of GPx5 mRNA was greater (P < 0.05) in the caput epididymis than in the remaining tissues during both seasons. This study demonstrated that season has an ambiguous influence on the antioxidant defense system in stallion reproductive tissues. Seasonal differences in the present study, however, indicate that the reproductive system of stallions adapts well to environmental seasonal changes.

Expression of hypoxia inducible factor 1α and antioxidant enzymes: Superoxide dismutases-1 and -2 in ischemic porcine endometrium.

The aim of this study was to determine how 60-min ischemia changes the expression of hypoxia inducible factor 1α (HIF-1α) and superoxide dismutases (SOD)-1 and -2 in selected regions of porcine uterine horns. The results showed that 60-min ischemia of the porcine uterus conducted at the mid-secretory estrous phase caused decreased HIF-1α and increased SOD-2 gene expression. Higher expression of SOD-2 suggests that this enzyme may play an important role in the suppression of HIF-1α accumulation in an ischemic endometrium.

Local transfer of testosterone and aromatase activity in the spermatic cord in wild boar/pig hybrids in short-daylight and long-daylight periods.

The local transfer of testosterone (T) and immunolocalization of cytochrome P450 aromatase (P450arom) in the spermatic cord vessels of ten male wild boar×domestic pig hybrids were examined in December (short-daylight period) and June (long-daylight period). Total T concentration was determined in the jugular vein (JV) and free T concentration was estimated in the common carotid artery (CA), branches of the testicular artery supplying the testis (TA) and epididymis (EA), as well as in testicular veins draining blood from the testis (TV) and spermatic cord (SV). P450arom was immunolocalized in the arterial and venous vessels of the spermatic cord. The concentrations of total T in the JV and free T in the CA did not differ between the examined periods. However, in December, free T concentrations in the TA and EA were higher (p<0.01-0.001) than in the CA. In June, free T concentration was higher (p<0.01) in EA than in CA and TA. The concentrations of free T in the TV and SV were higher (p<0.001) than in the JV regardless of the period. Also, free T concentration in the SV was higher (p<0.05) in June than in December. P450arom was expressed in all layers of the arterial and venous vessels of the spermatic cord. In June, the intensity of the P450arom staining was higher than in December. The results suggest that the local supply of the male reproductive organs with steroid hormones operate in the hybrids of wild boar×domestic pig. This supply includes the local transfer of testosterone and the P450arom action.

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods.

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n = 5) and short day (SD; 8L:16D, n = 5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A (120) and VEGF-A (164) and our results indicate that VEGF-A (164) was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p < 0.05) and NRP-1 mRNA expression (p < 0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.

Retrograde and local destination transfer of uterine prostaglandin E2 in early pregnant sow and its physiological consequences.

The local destination transfer of prostaglandin E2 (PGE2) from the uterine lymph to arterial blood supplying the ovary and its retrograde transfer to arterial blood supplying the uterine horn and the effect of additional delivery of PGE2 into the ovary on the secretion of steroid hormones was studied in early pregnant gilts. The injection of PGE2 under the perimetrium caused an increase (P<0.001) in PGE2 concentration in both uterine venous effluent and ovarian and uterine arterial blood. The infusion of PGE2 into the ovarian artery increased the concentration of progesterone in ovarian venous blood on day 13 of pregnancy during (P<0.05) and after (P<0.001) infusion, and on day 14 of pregnancy after infusion (P<0.01). In conclusion, local destination transfer of PGE2 from uterine lymph and venous blood to the ovary may affect luteal function, and retrograde transfer of PGE2 to the arterial blood supplying the uterus may contribute to the prevention of regressive changes of the endometrium in early pregnant gilts.

Season controlled reproduction of undomesticated animals.

The following article is a summary of research on the influence of season on the reproductive processes in undomesticated animals. The results presented below show: a/ an annual hormonal profile of domestic pig and wild boar crossbreed and the antioxidant blood system in the different seasons, b/ the possibility of gonadptropic hormone stimulation in chinchillas which are in diestrus or infertile, c/ the possibility of using bison's semen (collected post mortem from the epididymis) for cryoconservation.