Sperm sexing with density gradient centrifugation in dogs

Junior, Aguinaldo Francisco Mendes; Scott, Caroline; Sicherle, Carmen Cecília; Guaitolini, Carlos Renato de Freitas; Dell’Aqua, Camila de Paula Freitas; Malossi, Camila Dantas; Araújo-Junior, João Pessoa; Souza, Fabiana Ferreira de

doi:10.1016/j.anireprosci.2018.11.003

Cited by 14 publications

(10 citation statements)

References 40 publications

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“…Sperm plasma and acrosomal membrane integrity was assessed using propidium iodide (1.5 µM; P4170, Sigma-Aldrich, St. Louis, USA), FITC-PSA (0.2 ng/mL; L-0770, Sigma-Aldrich) and Hoechst 33342 (7 µM; 14533, Sigma-Aldrich), as previously described (Mothé et al, 2018); samples were incubated at 37 °C for 15 min in the dark. To evaluate phosphatidylserine membrane translocation, an Annexin V-FITC Kit I for apoptosis estimation was used (550475; BD Bioscience Pharmingen) with PI and Hoechst according to the manufacturer's instructions; the samples were incubated at 37 °C for 15 min in the dark.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…For caspase activity evaluation, a caspase 3/7-FITC kit (C10427, Molecular Probes Inc.) with PI and Hoechst was used according to the manufacturer's recommendations, and the samples were incubated at 37 °C for 25 min in the dark. To assess mitochondrial membrane potential, JC-1 (153 µM; T3168, Molecular Probes Inc.) was employed according to methods described previously (Mothé et al, 2018), with incubation for 10 min at 37 °C.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Effects of the cryopreservation process on dog sperm integrity

et al. 2020

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Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

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Section: Flow Cytometrymentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

Effects of the cryopreservation process on dog sperm integrity

et al. 2020

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“…The dielectrophoretic layer was made from glass and had an electrode pattern (3) which connected to the power generator (4) via electrical wire (5, red line). The microchannel layer was made from silicone and had a microchannel inside (6). the microchannel was connected to silicone tube (7), and the fluid was controlled by a peristaltic pump (8).…”

Section: Sorting Processmentioning

confidence: 99%

“…Moreover, the DNA staining may also cause abnormal development and genetic mutation [3,4,5]. Many alternative techniques such as antibody specific to sperm antigen, density gradient centrifugation, and albumin filtration have been demonstrated but inconsistent results, and therefore unpopular [6,7,8]. Thus, a novel method for reliable and economical sperm sexing must be developed.…”

Section: Introductionmentioning

confidence: 99%

Enrichment of bovine X-sperm using microfluidic dielectrophoretic chip: A proof-of- concept study

Wongtawan

Dararatana

Thongkittidilok

et al. 2020

Heliyon

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The microfluidic dielectrophoretic (MF-DEP) chip is a new, economical and readily-available technology that might be used to enrich X-sperm for increasing female offspring in dairy farms. In this study, we sought to develop an MF-DEP chip to enrich X bovine sperm. The MF-DEP chip was composed of an electrode attached to a glass slide and a microchannel made from polydimethylsiloxane. Sex-sorted sperm from flow cytometry were used to identify optimal electric field conditions while unsorted sperm were later tested for sorting efficiency. The results show that during dielectrophoresis some sperm attached to the electrode (called positive DEP; pDEP) whereas other moved away from the electrode (called negative DEP; nDEP). X and Y-sperm responded to dielectrophoresis differently depending on various factors such as buffers, voltages, and frequencies. We found that the condition 4 V 1 MHz significantly reduced (P < 0.05) the percentage of Y-sperm to nearly 30 and therefore enriched X-sperm. The sorting efficiency was dependent on buffer, bull, sorting cycle, flow rate, electrical voltage, and frequency. Notably, the best sorting buffer found in this experiment was the conducting buffer, but this buffer significantly reduced sperm viability and motility. Other sperm-friendly buffers, TRIS and mHTF, were also used, but could not enrich X-sperm. In conclusion, this is a proof of concept that the MF-DEP chip can be effectively used to enrich bovine X-sperm. However, more research must be performed particularly to find the best sorting buffer to effectively sex-sort sperm while providing high motility and sperm viability.

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“…The most widely used method for sperm sex sorting is fluorescence-activated cell sorting (FACS) 34 . Since its inception, sperm sorting has not only successfully been applied in various breeds of cattle but also in sheep, horses, pigs, dogs, bottlenose dolphins and gorillas [35][36][37][38][39][40][41][42][43] . Due to ethical reasons, the use of sexed sperm in humans is extremely limited and restricted to families with a history of X-linked diseases such as hemophilia or Duchenne's muscular dystrophy 44 .…”

mentioning

confidence: 99%

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting

Pirez

Steele

Reese³

et al. 2020

Sci Rep

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To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

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Sperm sexing with density gradient centrifugation in dogs

Cited by 14 publications

References 40 publications

Effects of the cryopreservation process on dog sperm integrity

Effects of the cryopreservation process on dog sperm integrity

Enrichment of bovine X-sperm using microfluidic dielectrophoretic chip: A proof-of- concept study

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting

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