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Vicksburg, MS, United States

Ferre L.B.,Instituto Nacional de Tecnologia Agropecuaria | Malik G.,Instituto Nacional de Tecnologia Agropecuaria | Aller J.F.,Instituto Nacional de Tecnologia Agropecuaria | Alberio R.H.,Instituto Nacional de Tecnologia Agropecuaria | And 2 more authors.
Small Ruminant Research

Adult male Llamas (Lama glama) (n = 6) were collected using a receptive female and a thermo-electric artificial vagina assembled with a polyethylene collection bag with the following objectives: (a) to develop a reliable and repeatable semen collection technique for Llama, (b) validate semen evaluation tests for Llama, and (c) to determine the effect of collection frequency and season on Llama semen characteristics. First, the semen and sperm variables were recorded and validated through their own repeatability. Semen collection intervals tested were: 1X/week for three weeks and 3X/week for another three weeks, the second collection period occurring after two weeks of sexual rest. The collection frequency of 3X/week significantly reduced (P < 0.05) the sperm viability, motility, concentration, Total Sperm/Ejaculate, Total Motile Sperm/Ejaculate and Total Live Sperm/Ejaculate, but improved Total Sperm/Week and Total Live Sperm/Week. All recorded variables were significantly higher during the summer in comparison to the spring with the exception of morphology abnormalities, volume, and viability. Also, the 1X/week versus the 3X/week semen collection frequencies did not produce a significant difference in the percent of total motile sperm/week. Based on semen and sperm characteristics evaluated herein, Llama semen collected in the summer was better (P < 0.05), with regard to the majority of the variables analyzed, than semen collected in the spring. © 2015 Elsevier B.V.. Source

Gosalvez J.,Autonomous University of Madrid | Lopez-Fernandez C.,Autonomous University of Madrid | Hermoso A.,Autonomous University of Madrid | Fernandez J.L.,Complejo Hospitalario Universitario runa | And 2 more authors.

Sperm DNA damage is one of the many factors that have been associated with male infertility. However, sperm DNA fragmentation (SDF) assessment has been challenging because protamines render the nuclear chromatin highly compacted. For those fish species having sperm with protamines, one hypothesis is that the less compacted DNA could be more vulnerable to iatrogenic damage when used in artificial reproduction. The present investigation included three objectives: 1) apply the Sperm Chromatin Dispersion (SCD) technique using the Halomax-SCD test kit (Halotech DNA, Madrid, Spain) for the rapid assessment of SDF in zebrafish ( Danio rerio); 2) assess the dynamic aspects of SCD to determine zebrafish sperm DNA longevity in both activated and unactivated samples; and 3) to analyze if the dynamic level of zebrafish sperm DNA fragmentation has any impact on fertility and embryo viability. The results demonstrate the use of the Halomax-SCD test for assessing SDF in zebrafish and are congruent with results of DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) and Comet Assay. The averaged SDF values derived from 10 individuals were low immediately after sperm motility activation (1.4%. ±. 0.96) compared to 15. min later (10.6%. ±. 9.96), with increasing and very high rates of SDF (r-SDF), 0.6% units/min. Although SDF did not significantly influence oocyte fertilization capacity, it significantly impacted later embryo development and overall reproductive success, i.e., fertility rates higher than embryo viability values. © 2014 . Source

Kjelland M.E.,Texas A&M University | Kjelland M.E.,Genetics and Biotech LLC | Romo S.,National Autonomous University of Mexico | Kraemer D.C.,Texas A&M University
Avian Biology Research

The use of reproductive technologies such as somatic cell nuclear transfer (SCNT) for avian species has been limited by the inability to visualise the pronucleus or pronuclei within the blastodisc or germinal disc region, respectively, primarily due to the opacity of the large, lipid filled yolk. The main objective in the present study was to assess a method for visualising and enucleating the avian ovum, a critical step in developing the capability for cloning birds. The method utilised in the present investigation was epi-fluorescence transmitted light (top-side UV) microscopy (EFTLM), also known as fluorescence/oblique or fluorescence/differential interference contrast (DIC) illumination, combined with vital staining and DNA visualisation techniques. The use of EFTLM combined with micromanipulation methods adapted from mammalian cloning procedures showed that the vitelline membrane of the avian ovum can be pierced and aspiration of the pronucleus, once visualised, can be performed without compromising the ovum's structure. Two approaches for domestic chicken ova collection, i.e., in vivo and in vitro, were utilised and ova recovery rates were compared. Based on a statistical analysis, i.e., Fisher's exact test, the results of the in vivo versus in vitro ovulation ova recovery methods were significantly different (P < 0.05), with the in vivo ovulation method yielding more viable intact ova. In conclusion, enucleating the avian ovum using EFTLM combined with vital staining, DNA visualisation, and micromanipulation techniques can be a feasible option for future avian cloning endeavours; although it will require further refinement to improve overall efficiency. Source

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