Intra- and interboar variability in flow cytometric sperm sex sorting

Alkmin, Diego Vilela; Parrilla, Inmaculada; Tarantini, Tatiana; Parlapan, Laura; Olmo, David del; Vázquez, J.M.; Rodríguez‐Martínez, H.; Roca, Jordi

doi:10.1016/j.theriogenology.2014.05.008

Cited by 10 publications

(14 citation statements)

References 30 publications

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“…Therefore, the effectiveness of the sexing technology in this species is not an issue [9]. However, the efficiency of the sex sorting procedure in the porcine species can be influenced by interboar and intraboar variability in the sortability of spermatozoa due to differences in the ability of ejaculates to exhibit welldefined X and Y peaks in the split on a flow cytometry histogram [10,11]. Alkmin et al [10], analyzing 67 ejaculates from different boars, found that around 15% failed to exhibit a well-defined split in the first ejaculate (bad sperm sorters; interboar variability).…”

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

“…However, the efficiency of the sex sorting procedure in the porcine species can be influenced by interboar and intraboar variability in the sortability of spermatozoa due to differences in the ability of ejaculates to exhibit welldefined X and Y peaks in the split on a flow cytometry histogram [10,11]. Alkmin et al [10], analyzing 67 ejaculates from different boars, found that around 15% failed to exhibit a well-defined split in the first ejaculate (bad sperm sorters; interboar variability). Analyzing five ejaculates from three of the bad sperm sorter boars, the percentage of the ejaculates not exhibiting a well-defined split ranged between 20% and 70% (intraboar variability) [10].…”

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

“…Alkmin et al [10], analyzing 67 ejaculates from different boars, found that around 15% failed to exhibit a well-defined split in the first ejaculate (bad sperm sorters; interboar variability). Analyzing five ejaculates from three of the bad sperm sorter boars, the percentage of the ejaculates not exhibiting a well-defined split ranged between 20% and 70% (intraboar variability) [10]. Such variability in pigs, unlike other species (dogs, horses) [12,13], is not influenced by the percentage of non-viable spermatozoa in the semen samples but is closely related to ejaculate sperm concentration.…”

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

“…Such variability in pigs, unlike other species (dogs, horses) [12,13], is not influenced by the percentage of non-viable spermatozoa in the semen samples but is closely related to ejaculate sperm concentration. Ejaculates are diluted to achieve the optimal sperm concentration for Hoechst 33342 (Ho) staining; during the staining step, samples from ejaculates with low sperm concentration would have a high proportion of seminal plasma that may alter Ho entrance into the sperm cell thereby affecting the effectiveness of DNA staining [10,11].…”

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

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Storage of sexed boar spermatozoa: Limits and perspectives

et al. 2016

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Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

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Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

Section: Factors Limiting the Large-scale Use Of Sexed Boar Spermatozoamentioning

confidence: 99%

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Storage of sexed boar spermatozoa: Limits and perspectives

et al. 2016

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“…To overcome the problems with spermatozoa variability among particular insemination doses (Alkmin et al 2014) it is necessary to have a wellestablished freezing and long-term storage protocol. In domestic animals a cryopreservation of cattle and horse semen is commonly used and improved (Barbas and Mascarenhas 2009), but the protocol for boar spermatozoa freezing is usually still based on egg-yolk extender (Pursel et al 1972).…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitination

Petelák¹,

Krylov²

2016

CZECH J ANIM SCI

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ABSTRACT:The present study is focused on the methodology of fluorescence activated cell sorting (FACS) of spermatozoa stained by the antibody against extracellular surface marker ubiquitin (eUb) and subsequent protocol for their long term storage in liquid nitrogen (LN). High level of spermatozoa surface ubiquitination has been previously discussed as a negative quality marker. From a general point of view, any other outer membrane antigen would be compatible with our approach. Regarding our experimental design we found that only those insemination doses with at least 40% of motile spermatozoa after freezing and thawing (F/T) in the egg-yolk medium with lactose are suitable for the subsequent antibody staining and FACS. The sorting rate was sufficient for the preparation of up to 20 spermatozoa aliquots for intracytoplasmic sperm injections (ICSI). Two significantly different groups with good freezability were prepared and stored in LN (0.73% contamination of spermatozoa with high eUb level in non-ubiquitinated group and reversely 6.65% spermatozoa without eUb in highly ubiquitinated group). Sperm viability after FACS varied from 11 to 28% regardless of the used media (P = 0.15). Required viability of F/T sorted spermatozoa was obtained by using Solusem ® extender as a load and collection medium. In this case 12% of viable spermatozoa with progressive motility in low eUb level group and 7% in high eUb level group (P < 0.05) were detected. Our approach allows obtaining sufficient number of viable spermatozoa for subsequent artificial fertilization by ICSI. This procedure could be used for a wide variety of spermatozoa sorting based on different surface markers.

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Seminal plasma affects sperm sex sorting in boars

Alkmin

Parrilla

Tarantini

et al. 2016

Reprod. Fertil. Dev.

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Two experiments were conducted in boar semen samples to evaluate how both holding time (24h) and the presence of seminal plasma (SP) before sorting affect sperm sortability and the ability of sex-sorted spermatozoa to tolerate liquid storage. Whole ejaculate samples were divided into three aliquots immediately after collection: one was diluted (1:1, v/v) in Beltsville thawing solution (BTS; 50% SP); the SP of the other two aliquots was removed and the sperm pellets were diluted with BTS + 10% of their own SP (10% SP) or BTS alone (0% SP). The three aliquots of each ejaculate were divided into two portions, one that was processed immediately for sorting and a second that was sorted after 24h storage at 15-17°C. In the first experiment, the ability to exhibit well-defined X- and Y-chromosome-bearing sperm peaks (split) in the cytometry histogram and the subsequent sorting efficiency were assessed (20 ejaculates). In contrast with holding time, the SP proportion influenced the parameters examined, as evidenced by the higher number of ejaculates exhibiting split and better sorting efficiency (P<0.05) in semen samples with 0-10% SP compared with those with 50% SP. In a second experiment, the quality (viability, total and progressive motility) and functionality (plasma membrane fluidity and intracellular generation of reactive oxygen species) of sex-sorted spermatozoa were evaluated after 0, 72 and 120h storage at 15-17°C (10 ejaculates). Holding time and SP proportion did not influence the quality or functionality of stored sex-sorted spermatozoa. In conclusion, a holding time as long as 24h before sorting did not negatively affect sex sorting efficiency or the ability of sorted boar spermatozoa to tolerate long-term liquid storage. A high proportion of SP (50%) in the semen samples before sorting reduced the number of ejaculates to be sorted and negatively influenced the sorting efficiency, but did not affect the ability of sex-sorted spermatozoa to tolerate liquid storage.

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Intra- and interboar variability in flow cytometric sperm sex sorting

Cited by 10 publications

References 30 publications

Storage of sexed boar spermatozoa: Limits and perspectives

Storage of sexed boar spermatozoa: Limits and perspectives

Cryopreservation of fluorescence activated cell sorted boar spermatozoa based on extracellular ubiquitination

Seminal plasma affects sperm sex sorting in boars

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