Metabolomic signature of spermatozoa established during holding time is responsible for differences in boar sperm freezability†.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.

Hydroxy-selenomethionine as an organic source of selenium in the diet improves boar reproductive performance in artificial insemination programs.

This study aimed to compare different selenium (Se) sources in the diet on boar's semen quality and fertility. For this, 28 boars aged 8 to 28 mo were fed with the following dietary treatments for 95 d: 0.3 mg Se/kg as sodium selenite (SS; n = 14) and 0.3 mg Se/kg as hydroxy-selenomethionine (OH-SeMet; n = 14). During this period, two experiments were carried out. In experiment 1, the semen of all boars was evaluated every 2 wk. Raw semen was initially evaluated for the processing of seminal doses, which were stored at 17 °C for 72 h, followed by sperm quality assessments. Furthermore, Se concentration and glutathione peroxidase (GPx) activity were measured in the seminal plasma. In experiment 2, 728 females were inseminated weekly with seminal doses from boars of the different experimental groups to further assess in vivo fertility and litter characteristics. Results demonstrated that boars fed OH-SeMet had more Se in their seminal plasma (P < 0.05), showing the greater bioavailability of the organic source in the male reproductive system. Moreover, boars fed OH-SeMet tended (P < 0.10) toward a higher total sperm count in the ejaculate (66.60 vs. 56.57 × 109 sperm) and the number of seminal doses (22.11 vs. 18.86; 3 × 109 sperm/dose) when compared with those fed SS. No effect of the dietary treatments was observed on GPx activity in seminal plasma (P > 0.05) as well as on raw and stored semen quality (P > 0.05). Under in vivo conditions, seminal doses from boars fed OH-SeMet tended (P < 0.10) toward a higher pregnancy rate at weeks 3, 5, and 8, and also resulted in a higher (P < 0.05) percentage of pregnant females in the overall period (99.30 vs. 97.00). In conclusion, the replacement of SS with OH-SeMet in boars' diet can improve sperm production and results in better reproductive performance for them, bringing greater productivity and profitability to artificial insemination centers and commercial pig farms.

The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols.

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h.

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation.

Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O2.-). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = l-arginine (10 mM - In); 3) L = L-NAME (1 mM - Ih); 4) M = methylene blue (100 mM - Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The l-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. l-arginine treatments improved acrosome reactions and differed from M and AM. NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.

Hyaluronidase administered with xylazine-tiletamine-zolazepam into adipose tissue shortens recovery from anesthesia in pigs.

OBJECTIVE: To evaluate the effect of hyaluronidase on uptake, duration and speed of elimination of xylazine-tiletamine-zolazepam administered in the subcutaneous fat over the dorsal lumbar region of swine. STUDY DESIGN: Blinded, randomized, crossover study. ANIMALS: Six healthy Landrace/Large White pigs weighing 132±24 kg (mean±standard deviation). METHODS: Animals were administered xylazine (1 mg kg-1) and tiletamine-zolazepam (8 mg kg-1) (control treatment, CON), or xylazine-tiletamine-zolazepam at the same doses with hyaluronidase (400 IU) (treatment HYA). The treatments were administered into the dorsal lumbar adipose tissue, 2.5-3.0 cm laterally from the spinous process of the second lumbar vertebra. The latency, anesthesia and recovery periods were measured. Heart rate, noninvasive systolic, diastolic, and mean arterial pressures, respiratory rate, hemoglobin oxygen saturation and rectal temperature were recorded every 10 minutes for up to 50 minutes. RESULTS: One animal in CON and one animal in HYA were responsive to stimulation and did not allow safe handling. No significant difference was found between treatments for latency (CON 11.3±5.9 minutes, HYA 7.4±5.1 minutes) and anesthesia (CON 53±53 minutes, HYA 49±38 minutes) periods. Recovery period was shorter in HYA (9±6 minutes) than in CON (32±16 minutes) (p < 0.05). Physiological variables were not significantly changed over time and were within accepted normal clinical limits for the species in both treatments. CONCLUSION AND CLINICAL RELEVANCE: Hyaluronidase (400 IU) administered into adipose tissue in pigs did not reduce the latency and duration of dissociative anesthesia, but was associated with faster recovery.

Regulation of neural stem cells by choroid plexus cells population.

The choroid plexus is a tissue on the central nervous system responsible for producing cerebrospinal fluid, maintaining homeostasis and neural stem cells support; though, all of its functions still unclear. This study aimed to demonstrate the niches of choroid plexus cells for a better understanding of the cell types and functions, using the porcine as the animal model. The collected material was analyzed by histology, immunohistochemistry, and cell culture. The cell culture was characterizated by immunocytochemistry and flow cytometry. Our results showed OCT-4, TUBIII, Nestin, CD45, CD73, CD90 positive expression and GFAP, CD105 negative expression, also methylene blue histological staining confirmed the presence of telocytes cells. We realized that the choroid plexus is a unique and incomparable tissue with different niches of cells as pluripotent, hematopoietic, neuronal progenitors and telocyte cells, which provide its complexity, differentiated functionality and responsibility on brain balance and neural stem cells regulation.