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Carvalho J.O.,University of Sao Paulo | Sartori R.,University of Sao Paulo | Dode M.A.N.,Laboratory of Animal Reproduction
Animal Reproduction | Year: 2014

Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry. Source

Carvalho J.O.,University of Sao Paulo | Silva L.P.,Laboratory of Mass Spectrometry | Sartori R.,University of Sao Paulo | Dode M.A.N.,Laboratory of Animal Reproduction | Dode M.A.N.,University of Brasilia
PLoS ONE | Year: 2013

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing. © 2013 Carvalho et al. Source

Kikuchi K.,Japan National Institute of Agrobiological Science | Nakai M.,Japan National Institute of Agrobiological Science | Kashiwazaki N.,Laboratory of Animal Reproduction | Kashiwazaki N.,Azabu University | Kaneko H.,Japan National Institute of Agrobiological Science
Animal Science Journal | Year: 2011

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early-stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro-matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science. Source

Bessa I.R.,University of Brasilia | Nishimura R.C.,University of Brasilia | Franco M.M.,University of Brasilia | Franco M.M.,Laboratory of Animal Reproduction | And 2 more authors.
Reproduction in Domestic Animals | Year: 2013

Contents: The aim of this study was to investigate the expression profile of candidate genes involved in competence during oocyte growth. The candidate genes (BMP15, OOSP1, H1FOO, H2A, H3A, H4, SLBP, DNMT1, DNMT3B, HAT1, HDAC2 and SUV39H1) were selected because of their possible involvement in determining oocyte developmental competence. Pre-antral and antral follicles were isolated from the ovaries of Zebu (Bos indicus) cows, measured and classified into the following categories according to their diameter: (i) oocytes from primordial follicles: diameter <20 μm, (ii) oocytes from primary follicles: 25-35 μm, (iii) oocytes from small secondary follicles: 40-60 μm, (iv) oocytes from large secondary follicles: 65-85 μm, (v) oocytes from small antral follicles: 100-120 μm, and (vi) oocytes from large antral follicles: >128 μm. Total RNA was extracted from four pools of 25 oocytes for each category of follicles, and the genes were quantified by qPCR. Target gene expression was normalized using the gene PPIA. The results suggest that stocks of the studied transcript genes accumulate before the final phase of folliculogenesis. The HDAC2 gene was the only gene in which a differential expression was observed at stage associated with competence acquisition. © 2013 Blackwell Verlag GmbH. Source

Gomes R.G.,Laboratory of Animal Reproduction | Andrade E.R.,Laboratory of Animal Reproduction | Lisboa L.A.,Laboratory of Animal Reproduction | Ciquini A.,Laboratory of Animal Reproduction | And 3 more authors.
Theriogenology | Year: 2012

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h. © 2012 Elsevier Inc. Source

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