Reverse transcriptase and protease inhibitors mutational viral load in HIV infected pregnant women with transmitted drug resistance in Argentina.

OBJECTIVE: Argentina has reported high levels of transmitted drug resistance (TDR), in HIV-infected pregnant women by population sequencing. We aimed to describe, in patients with TDR, the percentage of quasispecies harboring resistance mutations (RAMs) and mutational load (ML). METHODS: Retrospective study in a cohort of 40 naïve HIV-infected pregnant women, whose pretreatment samples had been genotyped by TRUGENE (period 2008-2014). Samples were re-sequenced with Ultra-deep Sequencing and ML was calculated considering baseline HIV-1 RNA load multiplied by the frequency of quasispecies harboring RAMs. RESULTS: TDR for NNRTIs, NRTIs and PIs was 17.5% (n=7 patients), 10% (n=4), 12.5% (n=5) respectively. Predominant NNRTI RAMs were K103N (n=4; 10%) and G190A/E/S (n=3; 7.5%). For NNRTIs, 78% of RAMs were present in >93.5% of viral population and ML was >1000 copies/mL (c/mL) for 89%, with a median (IQR) of 8330 c/ml (7738-29796). The following NRTI RAMs were described (per patient: % of quasispecies, ML): T215I (99.7%, 11014 c/ml); D67G (1.28%, 502 c/mL); M41L (79.8%, 88578 c/mL) and M184I (1.02%, 173 c/mL). Most frequent PI-RAMs were I85V, M46I, I50V and L90M (n=2, 5% each). For PIs, quasispecies with RAMs were <2.3% of viral population and ML was <350 c/mL for 77.8% of them. CONCLUSIONS: NNRTI-RAMs are predominant within the viral population, usually exceeding the threshold of 1000 c/mL, indicating potential higher risk of perinatal transmission. Conversely, PI mutations appear mostly as minority variants, with potential lower risk of transmission. Among NRTI, quasispecies harboring RAMs and ML values were variable.

Morphology, morphometry and chromatin distribution in llama sperm nuclei.

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 µm2 , perimeter 13.16 µm, length 5.12 µm, width 2.81 µm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

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)

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)

Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014-15.

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.

Efficacy of ultrasound-guided fine-needle aspiration performed by surgeons newly trained in thyroid ultrasound.

BACKGROUND: There are a growing number of thyroid ultrasound courses to train endocrinologists, pathologists, and surgeons to perform ultrasound-guided fine-needle aspiration (FNA). However, there are limited data to support the efficacy of ultrasound-guided FNA performed by nonradiologists. METHODS: We compared the efficacy of ultrasound-guided FNA performed by surgeons newly trained in thyroid ultrasound with that of the same technique performed by 1 experienced radiologist. The ratio of nondiagnostic examinations was used to compare the 2 groups. RESULTS: A total of 197 ultrasound-guided FNAs were performed on 172 women (95.53%) and 8 men (4.47%) over a 12-month period. The efficacy of ultrasound-guided FNA did not differ between groups. A nondiagnostic report was found in 23% and 25.7% of the examinations performed by the radiologist and surgeons, respectively. CONCLUSION: The efficacy of ultrasound-guided FNAs performed by our 2 surgeons was similar to that of our radiologist. © 2016 Wiley Periodicals, Inc. Head Neck 39: 439-442, 2017.

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.

Correlation and comparison of immunohistochemistry for HER2/neu, using the antibody SP3 and chromogenic in situ hybridization in breast carcinomas samples / Correlação e comparação de imuno-histoquímica para HER2/neu, utilizando o anticorpo SP3 com a hibridização in situ cromógena em amostras de carcinomas mamários

ABSTRACT Introduction: Advances in the field of molecular biology have provided the differentiation of molecular subtypes of breast tumors, providing better prognosis and important tools for the treatment of patients with breast cancer. Among these subtypes, the changes in the human epidermal growth factor receptor 2 gene (HER2/neu), increase its copy number and generating HER2 protein amplification. Studies show that patients with breast cancer HER2/neu amplified tend to relapse earlier and have shorter survival time, the monoclonal antibody Trastuzumab is the therapy indicated. The eligibility of patients for therapy is initially made by the immunohistochemistry (IHC) technique, which evaluates the expression level of the HER2 protein. After this evaluation, the cases with equivocal diagnosis (score 2+), are referred to a more accurate technique, the chromogenic in situ hybridization (CISH). Objective: To analyze the sensitivity and specificity of the antibody SP3, and determine their level of agreement with the CISH technique. Material and methods: Retrospective study in the database of the anatomy-pathology laboratory, in CISH tests reports for HER2/neu. Conclusion: The results revealed that clone SP3 showed 100% specificity and 92% sensitivity. IHC reveals variability in its results; however, it is known that the technique is an important tool in the daily routine of laboratories, contributing to the initial screening of patients with breast cancer, which later showed satisfactory results when compared with the CISH technique.

RESUMO Introdução: Avanços no campo da biologia molecular têm proporcionado a diferenciação dos subtipos moleculares das neoplasias mamárias, fornecendo melhor prognóstico e ferramentas importantes para a terapêutica de pacientes com câncer de mama. Entre esses subtipos, as alterações ocorridas no gene receptor tipo 2 do fator de crescimento epidérmico humano (HER2/neu) amplificam o seu número de cópias e geram o aumento da proteína HER2. Estudos mostram que pacientes portadoras de câncer de mama HER2/neu amplificado tendem a ter recaída mais cedo e tempo de sobrevida menor, sendo o anticorpo monoclonal Trastuzumab a terapia indicada. A elegibilidade das pacientes para a terapia é feita inicialmente pela técnica de imuno-histoquímica (IHQ), que avalia o nível de expressão da proteína HER2. Após essa avaliação, os casos que apresentam diagnósticos equívocos (escore 2+) são encaminhados para uma técnica mais precisa, a hibridização cromógena in situ (CISH). Objetivo: Analisar a sensibilidade e a especificidade do anticorpo SP3, além de determinar o seu nível de concordância com a técnica de CISH. Material e métodos: Estudo retrospectivo no banco de dados de um laboratório anatomopatológico, em laudos de exames de CISH para HER2/neu. Conclusão: Os resultados revelaram que o clone SP3 apresentou 100% de especificidade e 92% de sensibilidade. A IHQ revela variabilidade em seus resultados, porém é sabido que a técnica é uma importante ferramenta na rotina diária dos laboratórios, contribuindo na triagem inicial das pacientes portadoras de câncer de mama, que, posteriormente, mostram resultados satisfatórios quando comparados com a técnica de CISH.

Prevalence variation of colpocytological abnormalities in adolescents and adults in the years 2000 and 2010 / Variação da prevalência de anormalidades colpocitológicas em adolescentes e adultas nos anos de 2000 e 2010

Introduction: Cancer of the cervix is the second most common cancer among women worldwide and the frequency of precancerous lesions is increasing among adolescents. Objective: To study and compare the frequency of epithelial abnormalities in cervical cytology exams in adolescents and adults. Materials and methods: Retrospective study based on analysis of reports of cytology exams performed in the years 2000 and 2010 in adolescents (12-18 years old) and in adult women. The frequency of cytological alterations was explored, stratifying them into high-grade/carcinoma and low-grade lesions. Results: In 2000, rates of epithelial abnormalities were 3.08% in adolescents and 1.39% in adult females, and were 4.76% and 1.83% in 2010, respectively. The high-grade lesions/carcinomas were more common among adults in both years. Discussion: There was an increase in prevalence of epithelial abnormalities in the last decade, both in adolescents and in adults. Proportionally, the increase was higher among adolescents (6.7%) than in adults (2.2%). However, the rate of high-grade lesions/carcinoma was higher among adult women in the two analyzed years. Conclusion: It was observed that the frequency of cervical epithelial abnormalities was higher among adolescents than adults and increased between 2000 and 2010. .

Introdução: O câncer do colo do útero é o segundo tipo de câncer mais comum entre as mulheres em todo o mundo, e a frequência de lesões pré-neoplásicas vem aumentando entre as adolescentes. Objetivos: Estudar e comparar a frequência de anormalidades epiteliais em exames colpocitológicos em adolescentes e adultas. Materiais e métodos: Estudo retrospectivo com base na análise de laudos de exames colpocitológicos realizados nos anos de 2000 e 2010 em adolescentes (12 a 18 anos de idade) e em mulheres adultas. A frequência de alterações citológicas foi explorada, estratificando-as em lesões de alto grau/carcinoma e lesões de baixo grau. Resultados: Em 2000, as taxas de anormalidades epiteliais foram de 3,08% nas adolescentes e de 1,39% nas mulheres adultas; em 2010, de 4,76% e 1,83%, respectivamente. As lesões de alto grau/carcinoma foram mais frequentes entre as adultas em ambos os anos. Discussão: Tanto em adolescentes como em adultas, verificou-se aumento da prevalência de anormalidades epiteliais na última década. Proporcionalmente, o aumento foi maior entre adolescentes (6,7%) do que em adultas (2,2%). No entanto, a taxa de lesões de alto grau/carcinoma foi maior entre as adultas nos dois anos analisados. Conclusão: Observou-se que a frequência de anormalidades epiteliais cervicais é maior em adolescentes do que em adultas, tendo aumentado entre os anos de 2000 e 2010. .

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.

Llama oviductal sperm reservoirs: involvement of bulbourethral glands.

The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

In vitro production of embryos in South American camelids.

Studies in reproductive biotechnology techniques have been minimal in South American camelids (SAC). Complex reproductive characteristics of these species contribute to slow progress. Nevertheless, some techniques, such as in vitro fertilization, intracytoplasmic sperm injection and nuclear transfer have been applied and have produced advances in knowledge on embryo environment and in vitro conditions necessary for development. Embryo production may have a high impact in both domestic and wild camelids population. Studies addressed to improve in vitro embryo production and oocyte collection could be a potential key to develop IVF and embryo production as a routine procedure in camelids.

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.

In vitro embryo production in llamas (Lama glama) from in vivo matured oocytes with raw semen processed with Androcoll-E using defined embryo culture media.

The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.

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.

Morphometric analysis of llama (Lama glama) sperm head.

Llama production in Argentina has increased, as the international interest in breeding this type of animals has grown in the last years. Considering the great polymorphism that llama spermatozoa present at evaluation using light microscopy, the aim of this study was to objectively evaluate llama sperm head morphometry using digital morphometric analysis. Five ejaculates from each of eight males were obtained to evaluate morphometric parameters of 8000 sperm heads stained with Tinción 15(®). The following average results were obtained for each parameter: size parameters: area 20.09 µm(2), length 6.60 µm, width 4.14 µm, equivalent circle diameter 5.06 µm, curve length 5.79 µm and curve width 3.48 µm; boundary parameters: perimeter 18.54 µm and convex perimeter 17.34 µm; and shape parameters: roundness 1.28 and elongation 1.59. Morphometric parameters of sperm head were compared between ejaculates of the same male and between males. Significant differences between ejaculates of the same male were found for all parameters evaluated (P < 0.01). Significant differences between males were found for all morphometric parameters (P < 0.01) except for curve length, curve width and perimeter. The differences detected would indicate that there is not a single morphometric pattern for Lama glama sperm head, because parameter values cannot be standardised.

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.