Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen

Losano, João Diego de Agostini; Angrimani, Daniel de Souza Ramos; Pereira, Ricardo José Garcia; Rocha, A.M.; Criscuolo, T.; Barnabé, Valquíria Hyppólito; Barnabe, R. C.; Mendes, Clayton Quirino; Assumpção, M. E. O. A.; Nichi, Marcílio

doi:10.1111/and.12225

Cited by 17 publications

(13 citation statements)

References 54 publications

(57 reference statements)

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“…The inaccuracy of the CASA analysis can be due to the inability of the equipment to differentiate the spermatozoa bonded to the membrane from artifacts of the sperm slide, as the canine sperm has a small length (approximately 7 µm; DAHLBOM et al, 1997), which gendered the confused interpretation. The analysis of the sperm bonded to the perivitelline membrane using the CASA analysis system has been previously validated for the bovine species by Losano et al (2015).…”

Section: Discussionmentioning

confidence: 99%

“…Then, the membranes were extended on a microscopy slide and covered with 10 µL of the sperm DNA stain Ident® fluorescent probe (Hamilton Thorne Biosciences, Beverly, MA, USA), which remained at room temperature for 7 minutes (LOSANO et al, 2015). The membranes were covered with a coverslip and then submitted to computerized assessment of sperm motility.…”

Section: Sperm-binding Test To the Perivitelline Membrane Of Chicken mentioning

confidence: 99%

“…In fact, the improvement of reproductive biotechniques is essential for the preservation of genetic material from selected animals. However, the sperm quality for reproductive manipulations has to be guaranteed (LOSANO et al, 2015). Thus, semen needs to be accurately evaluated (AITKEN, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…However, such tests have reduced practical applicability for conventional sperm analysis laboratories, since they are expensive, laborious, and difficult to implement, and require specific equipment and materials. Thus, the sperm-binding to the perivitelline membrane of chicken eggs arises as an alternative test for sperm functional evaluation, as it allows an indirect and accurate analysis of the ejaculate fertilizing capacity (LOSANO et al, 2015). In other words, it can be carried out more frequently for seminal evaluation, being more accessible, more practical, and cheaper for dog breeders.…”

Section: Introductionmentioning

confidence: 99%

“…However, to our knowledge no studies regarding the standardization and evaluation of the efficiency of sperm-binding test to the perivitelline membrane of chicken eggs have been undertaken for canine sperm, mainly for post-thaw sperm samples. Conversely, tests have been performed for domestic birds, bulls, stallions, ram, boars, mice, and humans (ROBERTSON et al, 1997;BARBATO et al, 1998;WACLAWEK et al, 1998;AMANN et al, 1999;CORCINI et al, 2012;LOSANO et al, 2015). Thus, the aim of this study was to standardize and evaluate the efficiency of the sperm-binding test to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation.…”

Section: Introductionmentioning

confidence: 99%

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Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Brito¹,

Losano²,

Angrimani³

et al. 2017

Braz. J. Vet. Res. Anim. Sci.

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During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories. Keywords: Sperm cryopreservation. Egg yolk. Sperm analysis. Perivitelline membrane. Canine. ResumoDurante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convenci...

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Section: Discussionmentioning

confidence: 99%

Section: Sperm-binding Test To the Perivitelline Membrane Of Chicken mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Brito¹,

Losano²,

Angrimani³

et al. 2017

Braz. J. Vet. Res. Anim. Sci.

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Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

Arakaki

Salgado

Losano

et al. 2019

American J Primatol

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The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.

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Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

Arakaki

Salgado

Losano

et al. 2019

American J Primatol

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Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden-headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden-headed lion tamarins using two different exenders: BotuBOV ® (BB) and Test Yolk Buffer ® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm-egg-binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short-term measure for the conservation of golden-headed lion tamarins. K E Y W O R D S assisted reproductive technology, Callitrichidae, neotropical primates, semen extenders, sperm freezing

show abstract

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen

Cited by 17 publications

References 54 publications

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

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