Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests.

The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN2 vapors; automated: using a drop curve of -39.82 °C·min-1 for 113 s from -5 to -80 °C during the critical period; and subsequent immersion in NL2. Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.

Porcine Germ Cells Phenotype during Embryonic and Adult Development.

Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.

Initial Characterization of 3D Culture of Yolk Sac Tissue.

The role of the yolk sac (YS) in miscarriage is not yet clear, largely due to ethical reasons that make in vivo studies difficult to conduct. However, 3D cultures could provide a solution to this problem by enabling cells to be arranged in a way that more closely mimics the structure of the YS as it exists in vivo. In this study, three domestic species (porcine, canine, and bovine) were chosen as models to standardize 3D culture techniques for the YS. Two techniques of 3D culture were chosen: the Matrigel® and Hanging-Drop techniques, and the 2D culture technique was used as a standardized method. The formed structures were initially characterized using scanning electron microscopy (SEM), immunohistochemistry (IHC), and quantitative real-time PCR (RT-qPCR). In general, the 3D culture samples showed better organization of the YS cells compared to 2D cultures. The formed structures from both 3D methods assemble the mesothelial layer of YS tissue. Regarding the IHC assay, all in vitro models were able to express zinc and cholesterol transport markers, although only 3D culture techniques were able to generate structures with different markers pattern, indicating a cell differentiation process when compared to 2D cultures. Regarding mRNA expression, the 3D models had a greater gene expression pattern on the Hemoglobin subunit zeta-like (HBZ) gene related to the YS tissue, although no significant expression was found in Alpha-fetoprotein (AFP), indicating a lack of endodermal differentiation in our 3D model. With the initial technique and characterization established, the next step is to maintain the cultures and characterize the diversity of cell populations, stemness, functions, and genetic stability of each 3D in vitro model.

Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa.

This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 °C to the freezing lab. On arrival, samples were split into the treatments with the following additions before cryopreservation; 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Semen was evaluated for motility kinetics at 30, 120, and 240 min after thawing. Flow cytometry assessments were performed at 60 min after thawing. At all-time points evaluated, total and progressive motility were greater (P ≤ 0.05) in semen cryopreserved with GSH than in Control. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity; however, the proportion of capacitated spermatozoa were reduced (by -21.17%) in semen treated with BHT + GSH compared to Control (P ≤ 0.05). In contrast, there was a higher (P ≤ 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. For IVF, semen cryopreserved with both antioxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (-54.11%) compared to Control. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P ≤ 0.05) for BHT compared to Control. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment.

Impact of cryopreservation protocols (one- and two-step) on boar semen quality at 5 °C and post-thawing.

The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 109 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 109 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 109 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol.

Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions.

Culture conditions regulate the process of pluripotency acquisition and self-renewal. This study aimed to analyse the influence of the in vitro environment on the induction of porcine induced pluripotent stem cell (piPSCs) differentiation into primordial germ cell-like cells (pPGCLCs). piPSC culture with different supplementation strategies (LIF, bFGF, or LIF plus bFGF) promoted heterogeneous phenotypic profiles. Continuous bFGF supplementation during piPSCs culture was beneficial to support a pluripotent state and the differentiation of piPSCs into pPGCLCs. The pPGCLCs were positive for the gene and protein expression of pluripotent and germinative markers. This study can provide a suitable in vitro model for use in translational studies and to help answer numerous remaining questions about germ cells.

Copper and zinc hydroxychloride cosupplementation improve growth performance and carcass and reduce diarrhea frequency in grower-finisher pigs.

This study investigated copper (Cu) and zinc (Zn) hydroxychloride cosupplementation on the growth performance, diarrhea frequency, carcass, meat quality, and antioxidant activity in grower-finisher pigs. A total of 256 pigs were used from 70 to 154 days (d) of age, distributed in four treatments, with eight pigs in each pen and eight replications per treatment. Diets were provided to grower pigs from 70 to 112 days old and in the finisher, 112 to 154 days old. Copper was considered the low level at 100 mg Cu/kg and 90 mg Cu/kg, respectively, and 150 mg Cu/kg in both periods as high in the grower and finisher periods. In the grower and finisher period, zinc was cosupplemented in the diet at 80 mg Zn/kg and 70 mg Zn/kg, respectively. In the diets, T1 and T2 groups are the traditional inorganic sources for minerals (copper sulfate, CuSO4; zinc oxide, ZnO) and T3 and T4 hydroxychloride sources (copper hydroxychloride, CHC, and zinc hydroxychloride, ZHC). The flavomycin was associated with treatments with low Cu content in the inclusion of 50 g/ton. The experimental design was in randomized blocks, the data were submitted to analysis of PROC MIXED in SAS, the PDIFF test analyzed the treatment effect. At the finisher period, pigs fed both minerals from hydroxychloride source had a higher BW 154 d, average daily gain (ADG) 70 to 154 d, the hot and cold carcass weight and frequency of normal feces than those fed 150 mg Cu/kg and Zn from a traditional inorganic source (P < 0.05). The animals fed low Cu levels of the sulfate source had a higher ADG 70 to 154 d than those fed high Cu levels of the same source (P < 0.05). Pigs fed 150 mg Cu/kg cosupplemented with Zn from a hydroxychloride source had the highest carcass length (P < 0.05). There was no difference among the treatments for meat quality (P > 0.05). Pigs fed 150 mg Cu/kg and Zn from a traditional inorganic source had a higher superoxide dismutase (SOD) activity than the other treatments (P < 0.05). Animals fed low Cu levels from hydroxychloride had a higher malondialdehyde (MDA) formation than those fed sulfate source, regardless of the Cu levels and those fed high Cu levels of hydroxychloride (P < 0.05). In conclusion, 150 mg Cu/kg as copper sulfate cosupplemented to zinc oxide in the diet of growing and finishing pigs impairs the growth performance, carcass and increases diarrhea frequency, and copper and zinc hydroxychloride cosupplementation improves these characteristics.

Neural Derivates of Canine Induced Pluripotent Stem Cells-Like Cells From a Mild Cognitive Impairment Dog.

Domestic dogs are superior models for translational medicine due to greater anatomical and physiological similarities with humans than rodents, including hereditary diseases with human equivalents. Particularly with respect to neurodegenerative medicine, dogs can serve as a natural, more relevant model of human disease compared to transgenic rodents. Herein we report attempts to develop a canine-derived in vitro model for neurodegenerative diseases through the generation of induced pluripotent stem cells from a 14-year, 9-month-old female West Highland white terrier with mild cognitive impairment (MCI). Canine induced pluripotent stem cells-like cells (ciPSCLC) were generated using human OSKM and characterized by positive expression of pluripotency markers. Due to inefficient viral vector silencing we refer to them as ciPSCLCs. Subsequently, the ciPSCLC were subjected to neural induction according to two protocols both yielding canine neural progenitor cells (cNPCs), which expressed typical NPC markers. The cNPCs were cultured in neuron differentiation media for 3 weeks, resulting in the derivation of morphologically impaired neurons as compared to iPSC-derived human counterparts generated in parallel. The apparent differences encountered in this study regarding the neural differentiation potential of ciPSCLC reveals challenges and new perspectives to consider before using the canine model in translational neurological studies.

Altrenogest during early pregnancy modulates uterine glandular epithelium and endometrial growth factor expression at the time implantation in pigs.

This study evaluated the effects of supplying altrenogest from day 6-12 of pregnancy on the endometrial glandular epithelium, corpora lutea (CL) morphology, and endometrial and CL gene expression. A total of 12 crossbred females (Landrace × Large White) were used. The females were assigned to 4 treatments according to a random design with a 2 × 2 factorial arrangement, with two categories (sow or gilt) and two treatments (non-treated and treated with altrenogest). On day 6 of pregnancy, animals were allocated to one of the following groups: non-treated (NT, n = 6; 3 sows and 3 gilts), and (T, n = 6; 3 sows and 3 gilts) treated daily with 20 mg of altrenogest, from day 6-12 of pregnancy. All animals were euthanized on day 13 of pregnancy. All CLs were individually weighed, and their volume were determined. The endometrial glandular density (GD), mean glandular area (MGA), and vascular density (VD) were determined by histomorphometric and immunohistochemical analyses. Endometrium samples were collected and analyzed by qRT-PCR to evaluate the abundance of transcripts for VEGF and IGF-I. Females in the T group had higher MGA (P < 0.05) compared to the NT group. There was no effect of treatment on GD or VD for both experimental groups. Sows in the T group had augmented expression of IGF-I (P < 0.05). Progestagen had no detrimental effect on CL morphology. In conclusion, altrenogest improves the uterine environment during the peri-implantation period in pigs without compromising corpora lutea development.

The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. I: Protective effect of melatonin and butylhydroxytoluene on sperm function.

The addition of antioxidants to the cryopreservation medium has been shown to exert a positive effect on the quality of frozen-thawed sperm in different species. The objective of the present study was to evaluate the effects of supplementing the freezing medium with butylhydroxytoluene (BHT) and melatonin (MEL) in frozen-thawed pig spermatozoa. With this purpose, six ejaculates coming from six separate boars were cryopreserved in traditional freezing medium (i.e. lactose/egg-yolk/glycerol; Control) supplemented with 1.0 mM BHT (BHT-1), 2.0 mM BHT (BHT-2), 0.01 µM MEL (MEL-1) and 1.0 µM MEL (MEL-2). We evaluated sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidation of thiol groups, and levels of total reactive oxygen species (ROS), peroxynitrite and superoxide anion (·O2-). We also analysed total (TM) and progressive sperm motilities (PM), and kinetic parameters at post-thaw (T0, T30 and T60). The BHT-2 and MEL-2 groups presented higher viability and acrosome integrity, and lower levels of peroxynitrite, ·O2- and lipid peroxidation than the control (P < 0.05), whereas MEL-2 diminished the levels of total ROS (P < 0.05). TM and PM were not affected by the treatment, while, LIN and STR shows differences between experimental groups. In conclusion, the addition of BHT and MEL to cryopreservation medium diminishes oxidative and nitrosative stress markers, which has repercussions for the integrity of plasma and acrosomal membranes of frozen-thawed spermatozoa.

Shade availability on pasture does not affect semen characteristics of Brahman bulls (Bos taurus indicus).

Testicular degeneration by heat is the leading cause of infertility in bulls. Beef cattle are generally farmed under hot and humid conditions, and consequently, the thermotolerance of each breed must be considered in their natural environment. This study aimed to evaluate the reproductive characteristics of Brahman bulls maintained in the grazing system, with or without shadow availability. Ten Brahman bulls aging between 24 and 30 months were allocated in two different paddocks, with or without shadow availability. The heat tolerance test was performed on three non-consecutive typical summer days. The semen samples were collected at four times points in a 14 days interval. The climate conditions were monitored throughout the experiment; and clinical evaluation, testicular consistence and scrotal circumference were measured before every semen collection. In addition, semen was evaluated regarding volume, aspect, turbulence, motility, straight movement, sperm concentration, and morphological exam. The studied Brahman bulls showed a high thermolysis capacity, high heat tolerance, and no differences in semen quality were observed between groups.

A degeneração testicular causada pelo calor é a principal causa de infertilidade em touros. Bovinos de corte geralmente são criados em condições de calor e umidade, e, consequentemente, a termotolerância de cada raça deve ser considerada em seu ambiente natural. O presente trabalho teve como objetivo avaliar as características reprodutivas de touros da raça Brahman mantidos em sistema de pastejo, com ou sem disponibilidade de sombra. Dez touros Brahman com idades entre 24 e 30 meses foram alocados em dois piquetes diferentes, com ou sem disponibilidade de sombra. O teste de tolerância ao calor foi realizado em três dias típicos de verão não consecutivos. As amostras de sêmen foram coletadas em quatro momentos em intervalos de 14 dias. As condições climáticas foram monitoradas durante todo o período experimental; e a avaliação clínica, consistência testicular e a circunferência escrotal foram avaliadas antes de cada coleta de sêmen. Ainda, o sêmen foi avaliado quanto ao volume, aspecto, turbulência, motilidade, vigor, concentração espermática e exame morfológico. Os touros estudados da raça Brahman apresentaram alta capacidade de termólise, alta tolerância ao calor, e não foram observadas diferenças na qualidade do sêmen entre os grupos.

Generation of neural progenitor cells from porcine-induced pluripotent stem cells.

In this study, porcine embryonic fibroblasts (pEFs) were reprogrammed into porcine-induced pluripotent stem cells (piPSCs) using either human or mouse specific sequences for the OCT4, SOX2, c-Myc, and KLF4 transcription factors. In total, three pEFs lines were reprogrammed, cultured for at least 15 passages, and characterized regarding their pluripotency status (alkaline phosphatase expression, embryoid body formation, expression of exogenous and endogenous genes, and immunofluorescence). Two piPSC lines were further differentiated, using chemical inhibitors, into putative neural progenitor-like (NPC-like) cells with subsequent analyses of their morphology and expression of neural markers such as NESTIN and GFAP as well as immunofluorescent labeling of NESTIN, ß-TUBULIN III, and VIMENTIN. NPC-like cells were positive for all the neural markers tested. These results evidence of the generation of porcine NPC-like cells after in vitro induction with chemical inhibitors, representing an adequate model for future regenerative and translational medicine research.

Effects of different equilibration times at 5 °C on boar sperm cryotolerance.

Equilibration time (ET) is the period during which sperm cells are in contact with cooling/freezing media components at a temperature of 5 °C, providing a proper osmotic balance between the intra- and extra-cellular milieu. The present study aimed to determine the ET (0, 2, and 4 h) that results in greater post-thaw sperm quality and functions. Based on the post-thaw sperm membrane integrity and motility ratios, 20 ejaculates collected from five boars were classified as having good (GFE, n = 5) or poor (PFE, n = 15) freezing capacity. Ratios of post-thaw sperm with intact plasma membrane and acrosome were similar between ET (0 h: 37.02 % ± 2.85 %; 2 h: 34.59 % ± 7.12 %; 4 h: 37.87 % ± 4.44 %) in GFE samples. In PFE, ratios of sperm with intact plasma membrane and acrosome at post-thaw were greater (P < 0.05) after an ET of 2 h than after an ET of 0 h (2 h: 26.16 % ± 1.54 % and 0 h: 16.74 % ± 1.59 %). Also, ratios of post-thaw sperm with relatively lesser membrane lipids disorder were greater (P < 0.05) after an ET of 2 h than for other ET in both GFE (2 h: 21.97 % ± 4.24 % and 0 h: 16.63 % ± 2.38 %) and PFE (2 h: 16.65 % ± 1.40 % and 0 h: 13.23 % ± 1.25 %) samples. In conclusion, an ET of 2 h results in greater sperm cryotolerance in both GFE and PFE samples, which suggests that modifying the freezing protocol lead to an increase post-thaw sperm function and survival.

The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis.

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/ß were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/ß after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/ß. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability.

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-ß-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.

Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues.

INTRODUCTION: Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. OBJECTIVES: We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. METHODS: Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. RESULTS: Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. CONCLUSIONS: We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.

Does low-level laser therapy on degenerated ovine testes improve post-thawed sperm characteristics?

Low-level laser therapy (LLLT) can modulate redox state of the cell which could be useful to treat testicular degeneration and also prevent injuries by sperm cryopreservation. The aim of this study was to evaluate the effects of LLLT treatment on semen cryopreservation from rams submitted or not to testicular degeneration by testicular insulation. Eleven White Dorper rams were divided into four groups: animals that were not insulated (Control) and not treated (No Laser) (n = 2); animals that were not insulated and treated with LLLT (n = 3); animals that were insulated and not treated with LLLT (n = 3), and animals that were insulated and treated with LLLT (n = 3). Testicular insulation was performed using scrotal insulation bags for 72 h. LLLT treatment was 28 J/cm2 energy, 808 nm of wavelength, and 30 mW of power output, irradiated on testis for 15 days with an interval of 48 h. Three ejaculates from each ram were collected: before insulation, 23, and 59 days after insulation bag removal. Cryopreservation was performed of the third ejaculate. Sperm evaluation was performed before and after cryopreservation considering sperm motility, morphology, acrosomal and plasma membrane integrity, mitochondrial potential, and oxidative stress. As expected, cryopreservation had a negative effect on several sperm motility characteristics and sperm membranes. LLLT treatment did not improve sperm quality from rams submitted to testicular insulation. Thus, testicular insulation and cryopreservation effects on spermatozoa were not attenuated by LLLT in this study.

Removal of seminal plasma prior to liquid storage of boar spermatozoa: A practice that can improve their fertilizing ability.

Seminal plasma (SP) plays a vital role in the maintenance of sperm function and integrity along with being involved in their communication with the female reproductive tract. Under in vitro conditions, although it is generally accepted that boar semen is better preserved when SP is diluted (extended) or removed (cryopreserved), its role during storage is not completely elucidated. In this context, the current study sought to determine the role of SP during storage of boar spermatozoa at 17 °C for 72 h. Thus, two treatments were prepared with semen from the sperm-rich fraction (SRF) of boar ejaculate previous to storage in liquid state: 1) PSP: semen directly extended in Beltsville Thawing Solution (BTS), and 2) ASP: semen first centrifuged with subsequent removal of supernatant (containing SP) followed by suspension of sperm in BTS. From this, two experiments were conducted separately in this work: 1) in vitro and 2) in vivo assays. The former aimed to evaluate how sperm capacity responds to in vitro capacitation (IVC) and whether their quality is affected by previous exposure to SP. In the latter, the objective was to understand how important these previous conditions can be for reproductive performance after artificial insemination (AI). According to our results, the previous removal of SP does not affect sperm quality and the response of these cells to IVC (P > 0.05) along with resulting in a lower percentage of acrosome damage in them [12.87 ±â€¯0.76 (ASP) vs. 16.38 ±â€¯0.73 (PSP)] (P < 0.05). This improved preservation of acrosome integrity in the absence of SP can explain the higher fertility rate (%) [63.27 ±â€¯23.47 (ASP) vs. 38.57 ±â€¯16.30 (PSP)] and number of implanted embryos at 28 days after AI (13.71 ±â€¯4.88 (ASP) vs. 7.16 ±â€¯4.02 (PSP)] (P < 0.05) presented by gilts inseminated with seminal doses of ASP. In conclusion, removal of SP prior to liquid storage of boar sperm for 72 h can be beneficial for their fertilizing ability.