Effect of Atomized Black Maca (Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca (Vicugna pacos) Epididymal Spermatozoa.

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.

Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium.

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

Oocyte Selection for In Vitro Embryo Production in Bovine Species: Noninvasive Approaches for New Challenges of Oocyte Competence.

The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30-40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of noninvasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better competence. In addition, the combination of oocyte and zygote selection via brilliant cresyl blue (BCB) test, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in-vitro-derived technologies in livestock species.

Effect of a natural supplement containing glucosinolates, phytosterols and citrus flavonoids on body weight and metabolic parameters in a menopausal murine model induced by bilateral ovariectomy.

OBJECTIVE: To evaluate the effect of a herbal preparation containing glucosinolates, phytosterols and citrus flavonoids (supplement) on body weight and metabolic parameters usually impaired by menopause. METHODS: A pre-clinical experimental study carried out in twenty-five Swiss strain mice (Mus musculus) randomly distributed (1:1:1:1:1 ratio) to five groups to receive for ten weeks: (1) oral gelatinized maca extract 0.5625 mg/kg/day + bilateral ovariectomy (Maca + OVX); (2) oral supplement 0.5625 mg/kg/day + bilateral ovariectomy (S1 + OVX); (3) oral supplement 1.6875 mg/kg/day + bilateral ovariectomy (S2 + OVX); (4) oral saline 100 µl/kg/day + bilateral ovariectomy (OVX); and (5) oral saline 100 µl/kg/day + sham surgery (sham). The primary endpoint was change in body weight gain from baseline to final. Secondary endpoints were uterine weight and cholesterol, triglyceride, glucose, and glucose/triglycerides index values at the end of the study. A modified intention-to-treat analysis was performed through linear regression models and using the Bonferroni method to penalized p-values by multiple comparisons. RESULTS: Twenty-three animals completed the study. There was a significant average difference in weight gain, with a greater reduction in the S2 + OVX group compared to the OVX group (difference= -3.5; 95% CI (-5.27; -1.74); p < .001). S2 + OVX group also displayed a significant average reduction of total blood cholesterol (difference: -16.94; 95% CI (-33.73; -0.15); p = .037). No significant effects of the supplement were found on other secondary endpoints. CONCLUSION: In this murine menopausal model, triple oral supplement dose resulted in an average reduction of weight gain and total cholesterol levels, suggesting that the compound could have a potential effect at regulating menopausal altered metabolism.

Lateralization of the Dichotic Digits Test, Central Auditory Processes, and Evoked Potentials in Multiple Sclerosis.

Lateralization for central auditory processing (CAP) with dichotic digits recognition (DDR) test is believed expression of hemispheric dominance. Multiple sclerosis (MS) is considered an inflammatory and autoimmune alteration of central nervous system (CNS). Hearing alterations in MS and their role in CAP has not been well studied. A patient with MS and new kind of alteration in lateralization of CAP with DDR test is presented. A 53 year of age female with MS of 16 years of evolution, nine of them remained asymptomatic. She has a persistent advantage of the right ear for DDR test; but other monaural tests showed predominance of the left afferent pathway. Brainstem auditory evoked potentials (BAEPs) and long latency auditory evoked potentials (LLAEPs) showed adequate right response with deficits in organization of left response in BAEP, and N2 wave. In the contrary direction of previous publication, we disclosed advantage for DDR test, BAEP, and LLAEP in the right ear. We observed no left ear suppression; with predominance of correct left percentages in monaural psychoacoustics tests. We must keep on searching to find pathophysiological meaning of predominant of right or left auditory laterality as a CAP disorder in patients with MS.

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.

Myrciaria dubia "camu camu" flour as a magnetoprotector in male mouse infertility.

Possible adverse effects of extremely low frequency magnetic fields (ELF-MF) are linked to a decrease of antioxidant defenses and the production of free radicals. The increase of free radicals affects the mitochondrial functionality inducing apoptosis, which affects the phosphorylation and generation of key ATP in fertilization. Myrciaria dubia, known as "camu camu," is a fruit with high levels of ascorbic acid, which exerts an important antioxidant function in the prevention of premature cell damage. In this study, the effect of Myrciaria dubia flour on oxidative damage produced by ELF-MF (610 µT) was evaluated by detecting the activity of endogenous mitochondrial oxidoreductase enzymes in a complete sperm cycle of mice. We found that the MF caused a significant (P < 0.05) decrease in sperm quality, whereas the groups supplied with Myrciaria dubia flour (50 and 75 mg/kg of body mass) in ELF-MF exposure showed a significant recovery (P < 0.05) in parameters of viability, percentage of plasma membrane integrity and mitochondrial activity, and index of epidymal sperm. This suggests that Myrciaria dubia flour would have an antioxidant activity that counteracts the damaging effects of ELF-MF in spermatogenesis and could be used as a natural ELF-MF protector. Bioelectromagnetics. 40:91-103, 2019. © 2019 Bioelectromagnetics Society.

In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed with Dolichos biflorus by flow cytometry.

Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA-. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA- were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA- in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA- in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA-. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA- (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.

Disgerminoma puro de ovario en adolescente: presentación de un caso y revisión de la literatura / Pure Ovarian Dysgerminoma in a Teenager: Case Report and Literature Review

Las tumoraciones de ovario de naturaleza maligna son poco frecuentes en edades pediátricas. Los disgerminomas son tumores relativamente raros y constituyen alrededor del 2 % de todos los canceres ováricos, sin embargo, representan aproximadamente la mitad de los tumores malignos de células germinales. Se presenta el caso de una paciente de 14 años de edad, con antecedentes de displasia esquelética generalizada, que acudió con dolor abdominal y masa palpable en hipogastrio. Los exámenes de imagenología informaron que la tumoración estaba bien encapsulada y que presentaba aspecto sólido. Se asoció a la elevación de marcadores tumorales séricos por lo que se realizó excéresis del tumor y estudio anatomopatológico que concluyó el caso como disgerminoma puro del ovario derecho limitado al órgano. Su relativa escasa frecuencia nos permite comunicar el presente reporte de un disgerminoma en paciente en edad pediátrica portadora de una displasia esquelética.

Ovary tumor of malignant nature is infrequent in pediatric ages. Dyrgeminomas are not common and constitute around 2 % of all ovary cancers; however, they represent approximately half of the malignant tumors of germinal cells. A case of a 14-year-old patient is presented, with antecedents of skeletal generalized dysplasia, who presented to the doctor complaining of abdominal pain and a palpable mass in the hypogastrium. Imaging exams reported a well-encapsulated tumor with a solid aspect. It was associated to the elevation of seric tumor markers reason for which it was removed and the anatomic-pathologic study concluded the case as a pure right ovary dysgerminoma limited to the organ. It is relatively infrequent in a pediatric age patient carrier of a skeletal dysplasia so it is decided to be published.

Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation.

The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of alpaca sperm could be partially prevented by supplementation of the semen extender with 1 mM Tempo or Tempol.

Efecto antiinflamatorio y antioxidante del extracto hidroalcohólico de Petiveria alliacea

Los radicales libres y el daño oxidativo están relacionados con la muerte celular, siendo la peroxidación lipídica un mecanismo que lleva a la destrucción oxidativa de la membrana celular. Además, en procesos inflamatorios, uno de los primeros estadios es la permeabilidad vascular incrementada (formación del edema) seguido de la extravasación de los fluídos, migración de leucocitos al sitio dañado y liberación de proteínas pro-inflamatorias. Evaluamos el efecto antioxidante y antiinflamatorio para Petiveria alliacea “mucura”. Para el efecto antioxidante, se evaluó la formación de especies reactivas al ácido tiobarbitúrico como indicador de la peroxidación lipídica. Dosis 200mg/mL de Petiveria alliacea disminuyó significativamente en un 42% los niveles de MDA comparado con el agua (control negativo). En la evaluación antiinflamatoria, se indujo la inflamación por inyección de carragenina (solución al 1%), en la parte subplantar de ratones y en la “bolsa de aire subcutánea” de ratas para la inflamación aguda y crónica respectivamente. En la evaluación antiinflamatoria hay una máxima reducción del edema en un 23,26% a las 4 horas del tratamiento. Para la inflamación crónica hay una reducción del 25,9% y 29,5% del peso y volumen del exudado extraído, respectivamente, así como una reducción del 24% de peso de tejido fibroso. Estos resultados evidencian efecto antioxidante y antiinflamatorio de Petiveria alliacea

The free radicals and the oxidative damage are related with cellular death, with the lipid peroxidation being a mechanism that leads to oxidative destruction of the cellular membrane. Furthermore, in inflammatory process, one of the first stages is the incremented vascular permeability (formation of the oedema) followed with the extravasation of the fluids, migration of the leucocytes to the damaged site and release of pro-inflammatory proteins. Was evaluated antioxidants and anti-inflammatory of Petiveria alliacea “mucura”. For antioxidants effect, was evaluated formation of thiobarituric acid reactive substances as indicator of lipid peroxidation. Doses 200 mg/mL of Petiveria alliacea decrease significatly in 42% levels of MDA compared to water (negative control). In the antiinflammatory evaluation, inflammation was analyzed in the carrageenan-induced paw oedema test in mice (solution to 1 %) and in the "carrageenan-induced air-pouch formation" of rates for acute and chronic inflammation respectively. In anti-inflammatory evaluation there is a significant reduction of the edema in 23.26% at 4hrs at the treatment. For chronic inflammation there is a reduction of 25.9% and 29.5% of the weight and volume of exuded extract respectively, as well as a reduction of 24% of weight of fibrous tissue. These results demonstrate antioxidant and anti-inflammatory effects of Petiveria alliacea

A multiple stage approach for performance improvement of primary healthcare practice.

Chilean primary healthcare practice is analyzed using a Data Envelopment Analysis (DEA) multiple stage approach. We estimate the efficiency level of 259 municipalities nationwide. Since the efficiency score by itself is of limited value for decision making, we use a multivariate tool to help explain the effect of relevant factors. First, we use a cluster analysis to homogenize the units under study. Second, we use DEA to estimate the efficiency levels, which varies from 61% to 71% for urban municipalities, and from 51% to 56% in rural ones. Third, we use bootstrap to estimate confidence intervals for the efficiency scores, and a Biplot method to identify adequate variables to include in the Tobit Model, which is our last stage. We identify six factors associated with rural municipalities' operational efficiency, and two with urban ones. Knowing the efficiency level of municipalities can help determine ways to improve their efficiency.