The landscape of long noncoding RNA expression in the goat brain.

The brain regulates multiple metabolic processes, such as food intake, energy expenditure, insulin secretion, hepatic glucose production, and glucose and fatty acid metabolism in adipose tissue, which are fundamental for the maintenance of energy and glucose homeostasis during lactation and pregnancy. In addition, brain expression has a fundamental impact on the development of maternal behavior. Although brain functions are partly regulated by long noncoding RNAs (lncRNAs), their expression profiles have not been characterized in depth in any ruminant species. We have sequenced the transcriptome of 12 brain tissues from 3 goats that were 1 mo pregnant and 4 nonpregnant goats to investigate their lncRNA expression patterns. Between 4,363 (adenohypophysis) and 4,604 (olfactory bulb) lncRNAs were expressed in brain tissues, leading us to establish a set of 794 already annotated lncRNAs and 5,098 novel lncRNA candidates. The detected lncRNAs shared features with those of other mammals, and tissue-specific lncRNAs were enriched in brain development-related terms. Differential expression analyses between goats that were 1 mo pregnant and nonpregnant goats showed that the lncRNA expression profiles of certain brain regions experience substantial changes associated with early pregnancy (238 lncRNAs are differentially expressed in the olfactory bulb), but others do not. Enrichment analysis showed that differentially expressed lncRNAs from the olfactory bulb are co-expressed with genes previously linked to behavioral changes related to pregnancy. These findings provide a first characterization of the landscape of lncRNA expression in the goat brain and provides valuable clues to understand the molecular events triggered by early pregnancy in the central nervous system.

A protein-coding gene expression atlas from the brain of pregnant and non-pregnant goats.

Background: The brain is an extraordinarily complex organ with multiple anatomical structures involved in highly specialized functions related with behavior and physiological homeostasis. Our goal was to build an atlas of protein-coding gene expression in the goat brain by sequencing the transcriptomes of 12 brain regions in seven female Murciano-Granadina goats, from which three of them were 1-month pregnant. Results: Between 14,889 (cerebellar hemisphere) and 15,592 (pineal gland) protein-coding genes were expressed in goat brain regions, and most of them displayed ubiquitous or broad patterns of expression across tissues. Principal component analysis and hierarchical clustering based on the patterns of mRNA expression revealed that samples from certain brain regions tend to group according to their position in the anterior-posterior axis of the neural tube, i.e., hindbrain (pons and medulla oblongata), midbrain (rostral colliculus) and forebrain (frontal neocortex, olfactory bulb, hypothalamus, and hippocampus). Exceptions to this observation were cerebellum and glandular tissues (pineal gland and hypophysis), which showed highly divergent mRNA expression profiles. Differential expression analysis between pregnant and non-pregnant goats revealed moderate changes of mRNA expression in the frontal neocortex, hippocampus, adenohypophysis and pons, and very dramatic changes in the olfactory bulb. Many genes showing differential expression in this organ are related to olfactory function and behavior in humans. Conclusion: With the exception of cerebellum and glandular tissues, there is a relationship between the cellular origin of sampled regions along the anterior-posterior axis of the neural tube and their mRNA expression patterns in the goat adult brain. Gestation induces substantial changes in the mRNA expression of the olfactory bulb, a finding consistent with the key role of this anatomical structure on the development of maternal behavior.

The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters.

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

Soy lecithin as a potential alternative to powdered egg yolk for buck sperm cryopreservation does not protect them from mitochondrial damage.

The aim of this study was to address whether soy lecithin (SL) was an effective non-penetrating cryoprotectant for buck sperm cryopreservation in the presence of seminal plasma. There was also an attempt to determine the optimal concentration of BHT as an antioxidant in powdered egg yolk (PEY) or in SL based media. Two ejaculates were collected from six bucks and mixed ejaculates were aliquoted into washed, using centrifugation procedures, and unwashed samples. In Experiment 1, washed sperm were re-suspended in PEY (15%) or SL (1%) media, while unwashed semen was only diluted in SL medium. In Experiment 2, washed and unwashed sperm were diluted in PEY and SL media, respectively, with there being different BHT concentrations (0.6, 2.0 and 5.0 mM). In both experiments, after 4 h of refrigeration, there were no differences neither in sperm viability nor plasma membrane functional integrity (HOST) between groups when there were evaluations using eosin-nigrosine staining. After thawing, however, there was a negative effect on motility of washed sperm preserved in SL media. Furthermore, results from cytometry evaluations indicated there was a larger population of thawed sperm with intact plasma (SYBR-14+/PI-) and acrosome (PE-PNA-) membranes, but inactive mitochondria (Mitotracker deep red-) when SL media were used. When there was BHT supplementation, there was only a slight enhancement of motility of spermatozoa preserved in PEY media with 5 mM BHT. In conclusion, when effectiveness and efficiencies are considered, PEY is the non-penetrating cryoprotectant that should be utilized for buck sperm cryopreservation.

Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification.

The purpose of this study was to determine the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the open pulled Straw method (OPS). We evaluated the effects of pre-treating the oocytes with 1 microM Taxol on chromosome organization, spindle morphology, cortical granule distribution and the ability of fertilized oocytes to develop to the blastocyst stage. After calf or cow oocyte vitrification without Taxol, significantly higher proportions of spindle abnormalities in the form of abnormal spindle structures or dispersed or decondensed chromosomes were observed compared to fresh control oocytes. In contrast, when we compared calf oocytes pre-treated with Taxol before vitrification with control calf oocytes, similar percentages of oocytes showing a normal spindle morphology were observed. The percentages of oocytes with a peripheral cortical granule (CG) distribution increased when the oocytes were pretreated with Taxol and vitrified, while oocytes vitrified without Taxol pre-treatment gave rise to higher cortical distribution percentages. Cleavage and blastocyst rates were significantly lower for vitrified versus untreated oocytes, both in cow and calf oocytes. Significantly higher cleavage rates were obtained when calf and cow oocytes were vitrified with Taxol. Pre-treatment with Taxol before cow oocyte vitrification yielded significantly higher blastocyst rates. Calf oocytes, however, were unable to develop to the blastocyst stage, irrespective of previous Taxol treatment. These results indicate that the pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by the cryopreservation process, and potentially improves the subsequent development of vitrified bovine oocytes. Summary sentence: Pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and potentially improves the development of vitrified bovine oocytes.

Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments.

Studies were conducted to compare viability of immature and mature porcine oocytes vitrified in ethylene glycol (EG) using open-pulled straws (OPS). Oocytes that had been allowed to mature for 12 h (germinal vesicle group; GV) and 40 h (metaphase II group; MII) were divided into three treatments: (1) control; (2) treated with cytochalasin B and exposed to EG; and (3) treated with cytochalasin B and vitrified by stepwise exposure to EG in OPS. After warming, a sample of oocytes was fixed and evaluated by specific fluorescent probes before visualization using confocal microscopy. The remaining oocytes were fertilized and cleavage rate was recorded. Exposure of GV oocytes to EG or vitrification had a dramatic effect on spindle and chromosome configurations and no cleavage was obtained after in vitro fertilization. When MII oocytes were exposed to EG or were vitrified, 18 and 11% of oocytes, respectively, maintained the spindle structure and either EG exposure or vitrification resulted in substantial disruption in microfilament organization. The cleavage rates of mature oocytes after being exposed to EG or after vitrification were similar (14 and 13%, respectively) but were significantly less than that of control oocytes (69%). These results indicate that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII porcine oocytes had better resistance to cryopreservation than GV stage oocytes.

Expression of a green fluorescence protein-carrier protein into mouse spermatozoa.

Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7days to, at least, 16days after inoculation. The GFP/LGS was detected in the sperm heads after an "in vitro" fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins.