Seminal plasma affects the survival rate and motility pattern of raw llama spermatozoa.

The objectives of this study were to evaluate the effect over time of different percentages of seminal plasma (SP) on llama sperm characteristics in raw semen and correlate the techniques routinely used to evaluate sperm viability and acrosome status with the Fluorescein Isothiocyanate -Arachis hypogea agglutinin/Propidium Iodide (FITC-PNA/PI). Eighteen ejaculates, obtained from 6 male llamas using electroejaculation, were incubated in 0.1% collagenase in HEPES-TALP (HT), centrifuged and resuspended with SP and HT: 0, 10, 50 and 100% SP. Samples were incubated (37 °C) until evaluation at 0; 1.5 and 3 h. Split plot and factorial designs were used to analyze sperm motility, viability, membrane function and acrosome status and Spearman's test was used for correlation. At 0 h, samples with 100% SP showed oscillatory motility; whereas in samples with 0 and 10% SP, progressive motility was predominant. Viability, membrane function and total motility decreased significantly at 3 h of incubation in samples with 100% SP. Sperm with intact acrosomes were fewer in 0% SP media at all times. FITC-PNA/PI correlated with 6-Carboxyfluorescein Diacetate and Propidium Iodide (CFDA/PI) and with Coomassie Blue (CB) stains (r = 0.8; p = 0.0 and r = 0.5; p = 0.0 respectively). CONCLUSIONS: the motility pattern of llama sperm is influenced by the concentration of SP. The use of SP as the only medium is not able to maintain sperm motility, viability and membrane function for 3 h. A certain percentage of SP is necessary in the medium to avoid spontaneous acrosome reactions. The correlations observed could help to shorten evaluation times and reduce costs in sperm laboratories.

Bone marrow stem cell applied in the canine veterinary clinics / Células tronco de medula óssea utilizadas na clínica veterinária canina

Cell therapy represents an old therapeutic practice initiated with the transfusion of whole blood in different clinical situations. There is now a breakthrough in the study of multipotent stem cell therapy because of its functionality in regeneration of tissues, which promotes attention of the scientific community. Bone marrow is one of the main sources of multipotent stem cells, composed by hematopoietic stem cells responsible for the renewal of the cellular components of the blood, and mesenchymal stem cells that aid in the regeneration of tissues. These cells have a strong potential for the treatment of several diseases, due their main characteristics such as high plasticity, capacity for self-renewal and immunomodulation. Although, there are many studies that show good results with the use of cell therapy as a form of treatment for several diseases, some studies still show inconclusive or unsatisfactory results. Therefore, the objective of this study was to review the application of bone marrow stem cells in the canine model since improvements on the knowledge of the technique are necessary to enable its applicability with safety and efficacy.(AU)

A terapia celular representa uma antiga prática terapêutica iniciada com a transfusão de sangue total em diferentes situações clínicas. Atualmente há um avanço no estudo da terapia com células-tronco mesenquimais por conta de sua funcionalidade na regeneração de tecidos, o que promove uma crescente atenção do meio científico. A medula óssea é uma das principais fontes de células-tronco multipotentes, no qual se encontram as células-tronco hematopoiéticas, responsável pela renovação dos componentes celulares do sangue, e as células-tronco mesenquimais que auxiliam na regeneração de tecidos. Essas células têm um forte potencial para o tratamento de diversas enfermidades, uma vez que possuem como principais características alta plasticidade, capacidade de auto renovação e imunomodulação. Apesar de haver muitos trabalhos que apresentam bons resultados com a utilização da terapia celular como forma de tratamento para diversas enfermidades, alguns estudos ainda demonstram resultados inconclusivos ou não satisfatórios, por isso, objetivou-se com este trabalho revisar a aplicação das células-tronco derivadas da medula óssea no modelo canino uma vez que é necessário melhorias sobre o conhecimento da técnica para que possibilite a sua aplicabilidade com segurança e eficácia.(AU)

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium.

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze-thawing.

Comparison of differents methods of sperm selection of llama raw semen.

The objective of this study was to compare the efficiency of different sperm selection methods applied to the same llama ejaculate. Four treatments were compared: two variants of the swim up technique (with and without seminal plasma), and two different colloids, Androcoll-E-Large and Percoll(®). Using electroejaculation, 21 semen samples were obtained from 7 llama males (n=7, r=3). The ejaculates were incubated in a solution of 0.1% collagenase, to decrease thread formation, and then split into 4 aliquots: one aliquot was layered over a column of Androcoll-E-Large (SLC) and the second over a column of Percoll (45%). The third aliquot was deposited in a tube with culture medium and was incubated at a 45° angle for 30min at 37°C (SU1). The last aliquot was centrifuged to separate the spermatozoa and seminal plasma. The sperm pellet obtained was resuspended, and transferred to a tube with culture medium which was incubated at an angle of 45° for 30min at 37°C (SU2). Both aliquots SLC and P showed higher proportions of progressive motility and plasma membrane functionality (p≤0.05) than raw semen. There were no significant differences (p>0.05) in sperm viability and in normal spermatozoa between raw semen and treatments. Nevertheless, only SLC did not have a significant increase of bent tails. In conclusion SLC centrifugation would be the method of choice for selecting llama spermatozoa.

Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers.

UNLABELLED: The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3h at 38 °C with 5% CO2 and 100% humidity. TREATMENTS: Three aliquots were further incubated 1h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. CONTROLS: One without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2 ± 14.4% live+dead AR sperm) (P < 0.05). These samples showed no motility and viability was very low (0-30%). Both ionomycin and progesterone presented significantly higher (P < 0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live+dead sperm) and the acrosome-present CB pattern (r = 0.64; P = 0.000) in all the evaluated samples. CONCLUSIONS: the FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR.

Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa.

The aim of this study was to determine the effect of two equilibration temperatures (5 °C and room temperature) and two cryoprotectants (glycerol and dimethylformamide, both at 7%) on llama sperm cryopreservation. Llama ejaculates were divided into four aliquots. A lactose-EDTA-egg yolk (LEEY) extender with either 7% glycerol (LEEY-G) or 7% dimethylformamide (LEEY-DMF) was added to two of the aliquots, which were equilibrated for 20 min at room temperature and subsequently frozen. The other two aliquots were extended in LEEY, cooled to 5 °C, then LEEY-G or LEEY-DMF was added, equilibrated for 20 min at 5 °C and frozen. No significant differences (P > 0.05) were observed in membrane function and chromatin condensation between any of the freeze-thawing protocols. Post-thaw motility was greater (P < 0.05) in LEEY-DMF than LEEY-G. DNA fragmentation was not different between raw and frozen semen with LEEY-DMF but was high in all samples with glycerol. Our results indicate that 7% glycerol would be detrimental for llama spermatozoa, but further studies are needed to evaluate effectiveness if used at lower concentrations. Dimethylformamide preserved motility and DNA integrity of frozen-thawed llama spermatozoa and could be used to replace glycerol at the concentrations used in this study.

Development of an artificial insemination protocol in llamas using cooled semen.

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test.

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.

Evaluation of the effect of cooling and of the addition of collagenase on llama sperm DNA using toluidine blue.

The effect cryopreservation has on sperm chromatin condensation has been studied in many species but not in South American camelids. The objectives of this study were to evaluate with toluidine blue (TB) the effects of cooling and of adding collagenase on llama sperm DNA condensation. The optimum incubation time (30 s, 1.5 and 3 min) with a reducing agent (dithiothreitol) was also determined. When comparing cooled samples with the raw ejaculate, a significant increase in sperm showing a high degree of decondensation (TB positive) was observed (P = 0.005). A positive correlation was observed, both in raw and cooled semen, between sperm head morphological abnormalities observed in TB-stained cells and TB-positive sperm (highly decondensed DNA), but not with TB-intermediate spermatozoa (moderately decondensed DNA). No significant differences (P > 0.05) were observed in samples incubated with or without 0.1% collagenase. In cooled semen, but not in raw, a significant increase (P = 0.000) in reacted sperm (TB positive) was observed using 3-min incubation with 1% dithiothreitol (DTT). To conclude, cooling would seem to produce an increase in llama sperm chromatin decondensation. Also, 0.1% collagenase in H-TALP-BSA could be added to raw semen to aid its manipulation as it would not seem to increase DNA decondensation.

DNA content of Ovis musimon spermatozoa.

To our knowledge, the value of the haploid DNA content (C-value) of Ovis musimon (mouflon) has not been previously published. Therefore, the aim of the present work was to determine the C-value and the nuclear area of O. musimon sperm cells and compare both parameters with those of Ovis aries. Feulgen reaction, which is specific and stoichiometric for DNA, was carried out on semen smears. The C-value and sperm nuclear area were determined using microspectrophotometry and Gallus domesticus erythrocytes as standard species. The C-value of O. musimon was 3.02 ± 0.04 pg, and the sperm nuclear area was 23.92 ± 0.89 µm(2). The C-value and the sperm nuclear area of O. aries were 3.07 ± 0.03 pg and 22.98 ± 0.86 µm(2) respectively. The O. musimon C-value was not significantly different (P > 0.05) from that of O. aries, indicating that both species may have a very close phylogenetic relation.

Chromatin cytophotometric analysis of abnormal bovine spermatozoa.

Sperm head morphology is basically conditioned by the nuclear structure. The aim of the present work was to study the relation between nuclear morphological features, DNA content and chromatin distribution in morphologically normal vs. abnormal bovine spermatozoa. To this end, individual Feulgen-reacted spermatozoa were cytophotometrically studied. Chromatin compactation was evaluated by means of nuclear area, as well as mean and maximal absorbance of each nucleus. Morphological abnormality analysed included large, small, pear, narrow and round shapes, together with presumably 'diploid' sperms. Both large and small spermatozoa have a DNA content that does not differ significantly from normal values, but their area and mean and maximal absorbance are significantly different. Size variation seems basically due to altered chromatin compactation. The pear shapes have a narrower neck and a significant increase in maximal absorbance alone, which is invariably recorded in the neck zone whose increase would indicate a change in distribution and/or compactation. The narrow and round shapes fail to present significant variations in studied parameters. The possible 'diploids' differ significantly from normal cells in all studied variables, with a little area increase.