The successful production of "sterbel" hybrids using beluga (Huso huso) cryopreserved sperm.

This is the first report of the production of viable "sterbel" hybrids using cryopreserved beluga (Huso huso) sperm. In the present study, beluga cryopreserved sperm were used for fertilization and activation of eggs collected from four females of sterlet (Acipenser ruthenus). Sperm were cryopreserved with the use of a glucose methanol extender with the application of an automatic freezer. The mean sperm concentration of beluga was 1.54 × 109 mL-1. Cryopreservation did not significantly change sperm velocity and trajectory parameters (VCL, VSL, VAP, LIN, STR, ALH, BCF). Cryopreservation affected only the values of percentage of motile sperm (MOT) and progressive motility (PRG). The frozen/thawed sperm were subsequently used for the fertilization of the sterlet ova. The fertilized and activated eggs from each female were incubated in separate experimental incubation cages in the RAS system (at 15 °C). This experiment resulted in the production of inter-generic hybrids that were incubated until hatching. Experimental hybridization was characterized by 20-35% hatching rates and normal development of "sterbel" larvae. Experimental hybrids were verified using molecular and cytogenetic analyses. All produced specimens were characterized by sterlet maternal and beluga paternal genomes and were diploids with 120 chromosomes. This study presents the procedure of hybridization of the sterlet with beluga cryopreserved sperm that can be applied in sturgeon aquaculture or research focused on the biology of sturgeon reproduction.

First report on embryonic and larval development of 2n/3n mosaic sterlet.

Mosaicism is frequently observed in aquaculture practices, and it adversely affects the production as well as the restoration programme of the sturgeon. The purpose of the present study was the induction of 2n/3n mosaic in sterlet, Acipenser ruthenus L., and compare their embryonic and larval development with diploid control sterlet. Microsatellite DNA loci genotyping was conducted for the identification of the genotypes and parentage analysis. Embryonic development was monitored in experimental groups at every 24 h interval. Identification of individual stages of embryonic development was recorded based on a 36-degree scale of development. Additionally, the BW and body length (LT) of experimental fishes were taken during 110 days of the rearing period. The Fulton's condition coefficient (F), length-weight parameters, and specific growth rate (SGR) coefficient were calculated. The analysis of embryonic development of the 2n/3n mosaic and the diploid control group did not show differences. However, higher mortality (88%) was observed in 2n/3n mosaic groups in comparison to the diploid control groups (55%). BW and body length of 2n/3n mosaic sterlet were slightly lower than the diploid control sterlet, but the differences were not statistically significant. F analysis did not confirm a lower growth performance of the fishes in the 2n/3n mosaic group. Microsatellite DNA loci genotyping confirmed both the incidence of polyspermy and retention of the second polar body. This paper presents the first report on embryonic development and growth performance of 2n/3n mosaic sturgeons.

Profiling circulating microRNAs in the serum of pregnant and non-pregnant pigs reveals a plethora of reproductive status-dependent microRNAs.

Circulating, non-coding RNAs, such as microRNAs (miRNAs) have been proposed to be powerful pathophysiological indicators of pregnancy in animals and humans. Since their discovery, it is known that miRNAs can take part in numerous biological processes, including cell proliferation and differentiation during early embryonic development and establishment of pregnancy. Our recent studies have indicated that maternal blood can carry miRNAs reported previously at the embryo-maternal interface in pigs. To expand the scope of our research, we tested the hypothesis that miRNAs previously identified in conceptuses, trophoblasts, endometrium and uterine lumen-derived extracellular vesicles (EVs) collected before Day 20 of pregnancy can show reproductive status-dependent profiles in the serum of cyclic and pregnant crossbred pigs. Custom-designed TaqMan arrays, multiplex real-time reverse transcription (RT)-PCR and real-time RT-PCR allowed us to identify a number of reproductive status-dependent miRNAs in serum samples collected from pigs during the estrous cycle or pregnancy (Days 16 and 20). We found that serum samples were enriched with miRNAs involved in processes important during the estrous cycle and early pregnancy, e.g. cell sensitivity and viability, angiogenesis, embryonic cell proliferation and differentiation. Further validation revealed different abundance of ssc-miR-143-3p and ssc-miR-125b in pregnant and non-pregnant animals and correlation of ssc-miR-125b levels with litter size. In addition, analyzed serum samples contained both EVs and Argonaute2 proteins, which are known to be involved in miRNA transportation and intercellular communication. In summary, we identified several circulating miRNAs that differ in abundance between cyclic and pregnant animals and could serve as potential indicators of reproductive status in pigs during breeding management.

Expression of adiponectin and its receptors in the porcine hypothalamus during the oestrous cycle.

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2-3, 10-12, 14-16 and 17-19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14-16, whereas in SME, the most pronounced gene expression was found on days 2-3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17-19 and in POA on days 2-3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14-16. AdipoR1 gene expression in POA was potentiated on days 2-3 and 10-12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2-3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2-14 of the cycle), and they decreased on days 17-19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2-3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2-3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2-3 (p < 0.05), while in POA on days 17-19 and in SME on days 10-12 and 14-16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary.

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP - on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) - on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.

Expression of orexin A and B in the porcine hypothalamus during the oestrous cycle.

Orexins A and B are hypothalamic peptides engaged in a variety of physiological functions related to the control of energy homeostasis, sleep and wakefulness. The presence of orexin receptors in the tissues of the hypothalamus-pituitary-gonadal axis indicates that these hormones are also involved in the control of the reproductive system. The aim of this study was to compare the expression levels of prepro-orexin (a precursor of orexins A and B) mRNA in the porcine hypothalamic structures involved in reproductive processes - mediobasal hypothalamus (MBH), preoptic area (POA) and stalk median eminence (SME), during four stages (days 2-3, 10-12, 14-16, 17-19) of the oestrous cycle. In MBH, lower concentrations of PPO mRNA were observed on days 2-3 than in the remaining stages. In POA, the highest mRNA expression of PPO was noted on days 17-19. In SME, the highest concentrations of PPO was observed on days 2-3, and the lowest on days 14-16. We also investigated the intensity of OXA and OXB immunoreactivity and detected both peptides in all examined structures. In MBH, signal intensity for OXA was highest on days 14-16 and lowest on days 17-19. The highest levels of immunoreactivity were noted on days 2-3 and 10-12 in POA, and in SME additionally on days 17-19. OXB immunoreactivity in hypothalamic tissues also changed during the cycle, and the highest signal intensity was reported on days 10-12 in MBH, on days 14-16 in POA, and on days 14-16 and 17-19 in SME. The results of our study indicate that orexins A and B are produced in the porcine hypothalamus and that their concentrations vary subject to the pig's hormonal status. Our findings also suggest that orexins may affect reproductive functions at the highest level of the hypothalamus-pituitary-gonadal axis.

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine hypothalamus during the oestrous cycle.

Orexin A and B are hypothalamic peptides involved in the control of food intake, sleep patterns, autonomic and neuroendocrine systems. The biological actions of orexins are mediated via two G-protein coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). The present study analyzed mRNA and protein expressions of OX1R and OX2R in porcine hypothalamic structures engaged in GnRH production and secretion, preoptic area (POA), mediobasal hypothalamus (MBH), and stalk median eminence (SME) on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. The highest OX1R gene expression in POA was observed on days 17-19 of the cycle. Changes in the mRNA expression in MBH and SME throughout the cycle were negligible. The expression peak of OX2R gene in POA and SME occurred on days 17-19 as well. There were no changes in the gene expression in MBH. OX1R protein content in POA and SME also was the greatest on days 17-19 and OX2R protein expression was most pronounced in MBH and SME during the same phase of the cycle. In conclusion, fluctuation of OX1R and OX2R mRNAs and proteins content in pig hypothalamus dependently on the phase of the oestrous cycle suggests that orexins, through the influence on the hypothalamic-pituitary-ovarian axis, may affect reproductive functions.