Vicksburg, MS, United States
Vicksburg, MS, United States

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Ferre L.B.,Instituto Nacional de Tecnologia Agropecuaria | Bogliotti Y.,University of California at Davis | Chitwood J.L.,University of California at Davis | Fresno C.,National University of Costa Rica | And 3 more authors.
Reproduction, Fertility and Development | Year: 2017

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mMtheophylline (TH), 10mM caffeine and 5mMtheophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B.Taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval. © CSIRO 2017.


Ferre L.B.,Instituto Nacional de Tecnologia Agropecuaria | Bogliotti Y.,University of California at Davis | Chitwood J.L.,University of California at Davis | Fresno C.,Catholic University of Córdoba | And 3 more authors.
Reproduction, Fertility and Development | Year: 2016

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P<0.05) in cleavage rate (M199≤57%, SOF≤71% and TALP≤72%), blastocyst formation (M199≤9%, SOF≤20% and TALP≤19%), proportion of Grade 1 blastocysts (M199≤15%, SOF≤52% and TALP≤51%), proportion of Grade 3 blastocysts (M199≤58%, SOF≤21% and TALP≤20%) and hatching rates (M199≤29%, SOF≤60% and TALP≤65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199. © CSIRO 2016.


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 | Year: 2014

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.


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 | Year: 2015

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..


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.
Aquaculture | Year: 2014

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 .

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