Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig.

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3ß-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in vitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an in vitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.

Feline Adult Adipose Tissue-Derived Multipotent Stromal Cell Isolation and Differentiation.

Cats are among the most popular household pets. However, compared to other species, there is little information specific to feline adult mesenchymal stromal/stem cells. Despite the phylogenetic distance between domesticated cats, Felis silvestris catus, and humans, they share some similar health challenges like kidney disease, asthma, and diabetes. Investigative efforts have been focused on adult adipose-derived stromal/stem cell (ASC) therapies to address feline illnesses, including de novo pancreatic tissue generation for diabetes treatment. Given the relatively small size of domestic cats, optimized cell isolation from small quantities of adipose tissue is important in the development of feline ASC-based therapies. Additionally, there are unique features of feline ASC culture conditions and characterization. This chapter contains a few of the novel aspects of feline ASC isolation, culture, preservation, and differentiation.

Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

TLR7/8 agonist (R848) inhibit bovine X sperm motility via PI3K/GSK3α/ß and PI3K/NFκB pathways.

Sex-control technology have great economic value and is one of the hot topics in livestock research. To produce more milk, dairy farmers prefer female offspring. X/Y sperm separation is an effective method for offspring sex control. Currently, the major commercial production method for sperm separation is flow cytometry sorting in cattle. However, flow cytometry requires expensive equipment and long sorting times. So, a simple and inexpensive method for producing a higher number of dairy cows is required. In this study, R848 activates toll-like receptor 7/8 (TLR7/8), thereby separating X from Y sperm. The results showed TLR7/8 is expressed in the tail of X sperm. Immunofluorescence (IF) of testes, epididymis, and ejaculate shows that the number of TLR7+/8+ sperm cells is up to 50 %. Furthermore, TLR7/8 agonist (R848) affects mitochondrial function through the PI3K/GSK3α/ß/hexokinase and PI3K/NFκB/hexokinase signalling pathways, inhibiting X sperm motility, while the motility of Y-sperm remains unchanged. The difference in sperm motility causes Y sperm (with high motility) to move to the upper layer and X-sperm (with low motility) to the lower layer allowing the separation of X and Y sperm. Based on this study, we reveal a simple and effective method for enriched X/Y sperms from cattle.

Rules of thumb to obtain, isolate, and preserve porcine peripheral blood mononuclear cells.

One of the most used biospecimens in immunology are peripheral blood mononuclear cells (PBMC). PBMC are particularly useful when evaluating immunity through responses of circulating B- and T-cells, during an infection, or after a vaccination. While several reviews and research papers have been published aiming to point out critical steps when sampling, isolating, and cryopreserving human PBMC -or even analyzing any parameter before sampling that could impair the immune assays' outcomes-, there are almost no publications in swine research dealing with these topics. As it has been demonstrated, several factors, such as stress, circadian rhythmicity, or the anticoagulant used have serious negative impact, not only on the separation performance of PBMC, but also on the ulterior immune assays. The present review aims to discuss studies carried out in humans that could shed some light for swine research. When possible, publications in pigs are also discussed. The main goal of the review is to encourage swine researchers to standardize protocols to obtain, manage and preserve porcine PBMC, as well as to minimize, or at least to consider, the bias that some parameters might induce in their studies before, during and after isolating PBMC.

Measurement of canine Th17 cells by flow cytometry.

Th17 cells are T helper cells which play an important role during inflammation and autoimmune disease. To investigate the role of these cells in diseases in dogs in a clinical setting, methods for fast identification had to be established. Th17 cells are a rare cell population, for their measurement stimulation is recommended. To examine more samples simultaneously and to receive a relatively high purity of cell population of CD3 + CD4+ cells, different methods on various levels of preselection of cells as well as the possibility of storing blood overnight for measuring Th17 cells by flow cytometry were investigated. Firstly, to receive a high number of mononuclear cells, two different density gradients were compared and analysed. Furthermore, the enrichment of CD3 + CD4+ cells via depletion of CD8alpha+, CD11b + and CD21+ cells by cell sorting (autoMACS Pro Separator) was tested. It was also investigated whether stimulation processes led to better interpretation of results and whether there was a significant difference in measurement of directly processed blood samples and samples that had been stored overnight. In conclusion, the use of the density gradient (Lymph24+ Spin Medium) resulted in a purer cell population through a significant decrease in polymorphonuclear cells (*p = 0.01). After cell sorting, a significant difference in cell population purity was detected. Within the target fraction (containing mainly CD3 + CD4+ cells), CD8alpha+, CD21+, CD11b + cell percentages were significantly lower (***p < 0.001, *p < 0.02, ***p < .0001, respectively), and CD3 + CD4+ cell percentage was significantly higher (***p < .0001). There was a significant difference in Th17 cell percentage between unstimulated and stimulated cell populations (***p < .0001), but no significant difference in the percentage of unstimulated Th17 cells (p = 0.29) or stimulated Th17 cells (p = 0.71) in stored blood in comparison to directly processed EDTA blood samples. Finally, a modified protocol that offers an efficient way to investigate samples that were stored overnight by means of flow cytometry was evolved to research the role of Th17 cells in dogs with different diseases or in healthy populations.

Comparative transcriptome analysis reveals potential testosterone function-related regulatory genes/pathways of Leydig cells in immature and mature buffalo (Bubalus bubalis) testes.

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.

Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities.

Sheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests, SE are sensitized with an anti-sheep red blood cell stroma antibody. In presence of serum or plasma complement components, sensitized SE may initiate complement cascade activation via the classic pathway explored in the CH50 assay. Sensitized SE may also be used to prepare C3b-coated SE that, with the use of buffers favoring AP, are suitable for the C3 Nef hemolytic assay and for the hemolytic assay studying the AP decay activity of FH. In this chapter we describe how to prepare SE for these different hemolytic tests.

Manufacturing and banking canine adipose-derived mesenchymal stem cells for veterinary clinical application.

BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. AD-MSCs presented normal karyotype, and did not form in vivo tumors. No adverse events were noted in the case treated with intravenously AD-MSCs. CONCLUSIONS: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Small ruminant SexedULTRA™ sperm sex-sorting: Status report and recent developments.

Flow cytometry sperm sex-sorting based on the relative DNA difference between X- and Y-chromosome bearing populations is an established method that has allowed the production of pre-sexed offspring in a multitude of species and has been a commercial success in cattle production for the past twenty years. Several improvements to the technology and the processing methods have increased the sorting efficiency of ejaculates and the post-thaw quality of sex-sorted sperm, allowing for the fertility gap between conventional (non-sorted) and SexedULTRA™ sex-sorted sperm to be bridged. Small ruminant industries are now progressively testing sex-sorted sperm for application in their specific niches and environments. A review of the key advances and the recent developments in caprine, ovine and cervine sperm sex-sorting technology are described in this publication.

Methods in isolation and characterization of bovine monocytes and macrophages.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

Mouse Sertoli cells isolation by lineage tracing and sorting.

Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence-activated cell sorting (FACS). We bred the Amh-Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato-negative cells expressed α-smooth muscle actin (α-SMA), a peritubular myoid cell marker, but double-negative populations were also present. These findings suggest that vimentin lacks Sertoli cell-specificity and that α-SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α-SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Colloid centrifugation reduces bacterial load in chilled dog semen.

Conventional semen extenders contain antibiotics to prevent bacterial growth. Finding alternatives would be beneficial to minimize the development of bacterial resistance mechanisms. The aim of this study was to determine the effect of Single Layer Centrifugation (SLC) with Canicoll of dog semen on microbial load and sperm quality during cooled storage. Twenty-four ejaculates were obtained from healthy dogs by digital manipulation. Samples were diluted in Tris-citrate-fructose extender without antibiotics and divided into two treatment groups: SLC-selected samples and unselected samples. Sperm motility (CASA), viability and acrosome integrity (PI/FITC-PNA) as well as bacterial load of each microorganism species (colony-forming units/mL) were assessed at 0 and 48 h of storage at 4 °C. Results indicate SLC-selected dog spermatozoa have greater percentages of motility, viability and acrosome integrity (P < 0.05). Bacterial growth in SLC sperm samples was less (P < 0.05) than unselected samples. Removal of individual bacterial species varied from 91 % to 98 % for Escherichia coli (91.62 %), Streptococcus spp. (98.18 %), Staphylococcus spp.(95.33 %) and Pseudomonas spp. (92.50 %). In conclusion, the use of SLC with Canicoll has the potential to decrease bacterial load in chilled dog semen.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed paraffin-embedded chicken tissues using commercial antibodies.

Immunohistochemical method to detect avian lymphocytes is an efficient and reliable tool for accurate diagnosis, and immunological analysis of avian diseases. However, there are scarce studies reporting immunohistochemistry (IHC) using commercially available antibodies in formalin-fixed paraffin-embedded (FFPE) chicken tissues. In the present study, we established an immunohistochemical method to identify chicken T and B lymphocytes in FFPE chicken tissues using commercial antibodies against chicken or human antigens. For this IHC method, the five tested anti-T lymphocyte antibodies reacted with chicken T lymphocytes on the FFPE sections. Further, 10 commercial anti-B lymphocyte antibodies were tested; of these, three successfully detected chicken B lymphocytes for IHC. In particular, anti-human CD3 (clone F7.2.38) antibody was most suitable for the detection of chicken T lymphocytes, whereas anti-chicken B cell activating factor receptor (BAFF-R) antibody (clone 2C4) was most suitable for the detection of chicken B lymphocytes under our IHC staining conditions. These two antibodies reacted with numerous lymphocytes of all representative lymphoid tissues without problematic background staining and nonspecific reactions. Our results indicate that T and B lymphocytes in FFPE chicken tissues can be immunohistochemically detected using commercial antibodies.

Optimization of a rapid one-step platelet-rich plasma preparation method using syringe centrifugation with and without carprofen.

BACKGROUND: Carprofen and platelet-rich plasma (PRP) are widely used in small animal clinical practice. Separation layers have been used during blood centrifugation to increase platelet yield. The objectives of this study were to (1) identify the optimal centrifugation force for the one-step PRP preparation, (2) determine whether there is an advantage to using carprofen in one-step PRP preparation, and (3) compare platelet morphology from one-step PRP preparation with and without carprofen. We hypothesized that injectable carprofen (emulsion formula) could be used successfully as the separation layer in PRP preparation. RESULTS: Samples from 14 healthy dogs were used to determine the optimal centrifugation force using one-step PRP preparation in a disposable syringe without carprofen, with forces set at 300, 500, 700, 900, 1100, 1300, and 1500 xg for 5 min. Optimum centrifugation force, plasma volume, and platelet concentrations of one-step PRP preparation were found and recovered at 900 xg, 1.9 ± 0.28 ml, and 260.50 ± 58.39 X 103 cell/µl, respectively. Samples from 12 healthy dogs were used to determine the optimal force (with forces set at 300, 500, 700, and 900 xg) for 5 min using one-step PRP preparation with carprofen. Optimum centrifugation force, plasma volume, and platelet concentrations for one-step PRP preparation with carprofen were found and recovered at 500 xg, 0.62 ± 0.16 ml and 948.50 ± 261.40 X 103 cell/µl, respectively. One-step PRP preparation with carprofen increased the platelet yield from baseline by 1.76 and 4.95 fold, respectively. Samples from 3 healthy dogs were used to observe platelet morphologies after centrifugation by scanning electron microscopy. Images of platelets on glass slides from both preparation methods revealed pseudopods emerging from the margins of the discoid platelets. CONCLUSIONS: One-step PRP centrifugation both with and without carprofen increased the platelet yield, but using carprofen (emulsion formula) as a separation layer resulted in a higher platelet yield. The clinical usefulness of PRP products from these methods should be further investigated.

Isolation and Detection of Zika Virus-Infected Rhesus Macaques Lymph Node Cells and Splenocytes.

Zika virus (ZIKV) is a mosquito-borne viral infection that is shed in biological fluids promoting vertical and sexual transmission. Recent outbreaks of ZIKV have been associated with an increase in adult and fetal infection-related diseases. ZIKV infection in rhesus macaques is considered a robust animal model for studying Zika viral infection dynamics and fetal disease. A compelling feature of ZIKV is its ability to persist for long periods of time in immunocompetent hosts and during pregnancy, which may be linked to adverse infection outcomes. One consistent site of viral persistence is lymph node tissues. Utilizing this feature of ZIKV infection could be useful to diagnose viral persistence and to improve efficacy evaluation of antiviral vaccines and therapeutics, as well as for diagnostic and prognostic assessments in humans. We have developed a protocol to isolate lymph node cells using cell type-specific antibody-magnetic bead techniques followed by a one-step qRT-PCR detection of Zika virus RNA. This method fostered the identification of dendritic cells, macrophages, and B cells from the lymph node and spleen as harboring persistent ZIKV RNA.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

Isolation and culture of epithelial cells from stored buffalo semen and their use for the production of cloned embryos.

The aim of the present study was to isolate somatic cells from semen, a non-invasive source of donor somatic cells, for somatic cell nuclear transfer (SCNT) experiments. The study had two parts: (1) isolation and culture of somatic cells from semen, which was stored at 4°C; and (2) investigating the SCNT competence of semen-derived somatic cells. We successfully cultured somatic cells from freshly ejaculated semen, which was stored for different times (0, 4, 12, 24, 72 and 144h after semen collection) at 4°C, using a Percoll gradient method. Up to 24h storage, 100% cell attachment rates were observed; cell attachment rates of 66% were observed for the 72 and 144h storage groups. The attached cells observed in all groups examined were proliferated (100%). Cultured cells exhibited epithelial cell morphology and culture characteristics, which was further confirmed by positive expression of cytokeratin 18, an epithelial cell-type marker. We compared the SCNT competence of semen-derived epithelial cells and skin-derived fibroblasts. The cleavage rate, blastocyst production rate, total number of cells in blastocysts and the apoptotic index of blastocysts were similar for embryos produced from semen-derived epithelial cells and skin-derived fibroblasts, indicating that semen-derived epithelial cells can serve as donors for SCNT experiments. In conclusion, we demonstrate a method to culture epithelial cells from stored semen, which can be used to produce cloned embryos of breeding bulls, including remote bulls.

Spermatogonial stem cells identified by molecular expression of PLZF, integrin ß1 and reactivity to Dolichos biflorus agglutinin in alpaca adult testes.

The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty-four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica-Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin ß1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA+ ) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA+ ) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca.