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Burroughs C.A.,Colorado State University | Graham J.K.,Colorado State University | Lenz R.W.,Sexing Technologies | Seidel G.E.,Colorado State University
Theriogenology | Year: 2013

The objective was to determine which characteristics of bovine ejaculates affected efficacy of sex sorting bovine sperm by flow cytometry. The effects of first versus second ejaculates, seminal plasma content, addition of BSA, and seminal plasma from different bulls during staining were all studied, as was the effect of 8-hour storage with and without seminal plasma. Semen collected by artificial vagina was centrifuged at 1000 × g for 15 minutes to separate sperm from seminal plasma; seminal plasma was clarified by 10 minutes of additional centrifugation at 2000 × g. Sperm were rediluted to 160 × 106 sperm per mL with: Tyrode's medium plus albumin, lactate, and pyruvate (TALP) containing 0%, 5%, 10%, or 20% homologous seminal plasma, TALP containing 10% heterologous seminal plasma, or TALP containing 0.3% (control), 0.6%, or 1.2% BSA. After incubation with Hoechst 33342 for 45 minutes, an equal volume of TALP containing red food dye was added, and sperm were analyzed by flow cytometry/cell sorting to determine percent of live-oriented sperm, X sort rate, percent of membrane-impaired sperm, and split (degree of separation between X- and Y-bearing sperm populations). The percent of live-oriented sperm was higher for sperm incubated with 0% seminal plasma (64%) than for sperm incubated with 5%, 10%, or 20% seminal plasma (60%, 59%, and 58%, respectively; P < 0.05). The X sort rate was higher for sperm incubated with 0% seminal plasma than sperm with 20% seminal plasma (4.26 vs. 3.61 × 103 sperm per second). When seminal plasma was exchanged between bull ejaculates, only one bull had seminal plasma that was detrimental to sperm, resulting in 31% membrane-impaired sperm compared with a range of 16% to 19% for seminal plasmas from other bulls (P < 0.05). The addition of BSA did not affect sort efficiency at the concentrations studied. Sperm from six bulls stored for 8 hours without seminal plasma had more membrane-impaired sperm (which were discarded) during sorting (28%) than with seminal plasma (19%; P < 0.01), but higher postthaw motility postsorting (63%) than with seminal plasma (52%; P < 0.05). In conclusion, the presence of seminal plasma during staining and sorting decreased sort rates and percent of live-oriented sperm, and storing sperm without seminal plasma increased postthaw motility. © 2013 Elsevier Inc.

Corrales F.J.,Research Center Medica Aplicada | Mora M.I.,Research Center Medica Aplicada | Pello-Palma J.,University of Oviedo | Moreno J.F.,Sexing Technologies
Journal of Proteome Research | Year: 2013

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes. © 2013 American Chemical Society.

Gonzalez-Marin C.,Sexing Technologies | Gosalvez J.,Autonomous University of Madrid | Roy R.,Autonomous University of Madrid
International Journal of Molecular Sciences | Year: 2012

Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Underwood S.L.,University of Sydney | Bathgate R.,University of Sydney | Pereira D.C.,Sexing Technologies | Castro A.,Sexing Technologies | And 3 more authors.
Theriogenology | Year: 2010

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm. © 2010 Elsevier Inc. All rights reserved.

Pena F.J.,University of Extremadura | Macias Garcia B.,University of Extremadura | Samper J.C.,Sexing Technologies | Aparicio I.M.,University of Extremadura | And 2 more authors.
Theriogenology | Year: 2011

We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade. © 2011 Elsevier Inc..

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