Effect of Post‐Thaw Addition of Seminal Plasma on Motility, Viability and Chromatin Integrity of Cryopreserved Donkey Jack (<i>Equus asinus</i>) Spermatozoa

Sabatini, C; Mari, Gaetano; Mislei, Beatrice; Love, C.C.; Panzani, Duccio; Camillo, Francesco; Rota, Alessandra

doi:10.1111/rda.12419

Cited by 15 publications

(16 citation statements)

References 35 publications

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“…The average value of acrosome membrane integrity measured by fluorescence probes was lower than that reported in the literature for donkeys, an average of 44.5 % (Sabatini et al 2014). However, it is important to note that the average of alive sperm without acrosome reaction was similar to plasm membrane integrity.…”

Section: Discussioncontrasting

confidence: 57%

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Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

Oliveira¹,

Teixeira²,

Pignataro³

et al. 2017

PHK

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Summary: Reproductive biotechnologies such as cooling and freezing semen are employed in order to obtain genetic enhancement in domestic animal breeds. However, cryopreservation of donkey semen, as in several other domestic mammalian species, has not yet reached a standard procedure that provides satisfactory and repeatable results, as in bulls. Despite there are over 43 million donkeys worldwide, there are only few studies about donkey semen preservation. The aim of this study was to evaluate the predictive potential of hypo-osmotic test and the supra vital staining in raw semen to be used as indicators for donkey semen freezability. Ejaculates were collected twice per week from 10 fertile donkey stallions. Volume, appearance (color and density), concentration, total motility, morphology, sperm viability (supra vital staining -eosin-nigrosin-EN) and plasma membrane integrity (hypo-osmotic swelling test -HOST) were evaluated in raw semen. After dilution the samples were centrifugated and sperm pellets were resuspended in a freezing extender to a concentration of 50 ×10 6 cells/mL. Aliquots were packed into 0.5 mL straws and placed in the refrigerator (5°C) for 20 min. Subsequently these straws were kept above liquid nitrogen for 20 min, then plunged into nitrogen and stored in a holding tank. Post-thaw analyzes consisted in computerized analysis of sperm movement characteristics, sperm viability (EN), plasma membrane integrity (HOST and fluorescent probe) and acrosome membrane integrity using fluorescent probe. High correlation was found between EN (0.93) and HOST (0.69) in raw semen when compared with total motility after frozen-thawed semen. Low correlation was found between EN and HOST when compared with plasma and acrosomal membrane integrity in post-thawed with fluorescent probes. Since sperm motility is one of the key parameters to evaluate the quality of frozen semen, hypo-osmotic test and supra vital staining can be considered good predictive indicators in raw semen for donkey semen freezability.Keywords: Equus Asinus, cryopreservation, eosin-nigrosin, plasma membrane integrity, semen, reproduction Citation: Arruda de Oliveira R., Batista Silva Teixeira A., Almeida Pignataro T., Freitas M. L., Castro Alves Teixeira H., de Oliveira Carvalho J., Alves Nune Dode M., Pivato I., Budik S. (2017) Evaluation of Hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability. Pferdeheilkunde 33,[159][160][161][162][163][164]

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Section: Discussioncontrasting

confidence: 57%

“…But the progressive motility values (PROGMOT); linearity (LIN); average path velocity (VAP); progressive linear velocity (VSL) and curvilinear velocity (VCL) were lower than those reported by Sabatini et al (2014) being 40.1± 8.8 % PROGMOT; 110.0 ± 7.2 VAP; 100.4 ± 6.3 VSL and 160.8 ±13 VCL (Table 2). …”

Section: Discussionmentioning

confidence: 70%

Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

Oliveira¹,

Teixeira²,

Pignataro³

et al. 2017

PHK

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“…Thus, it is crucial to choose a suitable time scale for our study. Previous reports in donkeys [19] and horses [12] have shown that significant changes in sDF between treatments and individuals occurred from 6 to 24 h of incubation at 37 °C when using a chromatin dispersion test, or 4 h of incubation for the sperm chromatin structure assay (SCSA) by flow cytometry [20]. However, since one of the objectives of this study was to fine-tune a model, in order to adjust the model to reality, we needed to have as many points as possible.…”

Section: Discussionmentioning

confidence: 99%

New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models

Ortiz

Dorado

Morrell

et al. 2017

J Animal Sci Biotechnol

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BackgroundSperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen.ResultsAfter submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R2) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC.ConclusionSLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

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“…llama [54] or pig [55], addition of seminal plasma to the thawed sperm before insemination seems to improve fertilisation rate. In donkeys, the addition of seminal plasma to post-thaw sperm showed no improvement in in vitro sperm characteristics [27] and, as shown in our study, had no effect on pregnancy rate, when it was added to the thawed sample before insemination. This is yet another demonstration of the fact that species are different from each other and thus require species-specific customisation of protocols.…”

Section: Discussionmentioning

confidence: 63%

“…While much has been done and written on cryopreservation of stallion semen, only a handful of studies could be found in the scientific literature on issues related to donkey semen cryopreservation. These includes reports on several donkey breeds such as the Baudet Du Poitou [10–14], Zamorano-Leonés [15,16], Pêga [17–19], Martina Franca [20,21], Catalonian [13,22], Grand Noir du Berry [13], Andalusian [23–25], Amiata [26,27], Abyssinian (Lemma et al, unpublished data), the American standard breed [28], and recently also for the onager [29,30]. Post-thaw sperm quality in these studies, as judged by laboratory evaluation techniques, was fair to excellent.…”

Section: Introductionmentioning

confidence: 99%

Follicular size predicts success in artificial insemination with frozen-thawed sperm in donkeys

et al. 2017

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In asses, semen collection, cryopreservation, and artificial insemination (AI) with frozen-thawed semen have been scarcely described and success rate, particularly following AI, is reportedly low. In the absence of reliable protocols, assisted reproductive technologies cannot support the conservation efforts aimed at endangered wild ass species and domestic donkey breeds. Two experiments were conducted in this study. In experiment 1 we evaluated freezing Abyssinian donkey (N = 5, 4 ejaculates each) spermatozoa using three freezing extenders (Berliner Cryomedium + glycerol, BC+G; BotuCrio, BOTU; INRAFreeze, INRA) and two cryopreservation techniques (liquid nitrogen vapour, LNV; directional freezing, DF). Post-thaw evaluation indicated that BOTU and INRA were similar and both superior to BC+G (P ≤ 0.004 for all motility tests), and that DF was superior to LNV (P < 0.002 for all evaluation parameters). In experiment 2, relying on these results, we used Abyssinian donkey sperm frozen in BOTU and INRA by DF for AI (N = 20). Prior to AI, thawed samples were diluted in corresponding centrifugation media or autologous seminal fluids at 1:1 ratio. No difference was found between BOTU and INRA or between the addition of seminal fluids or media, all resulting in ~50% pregnancy, and no differences were noted between males (N = 4). The size of pre-ovulatory follicle was a significant (P = 0.001) predictor for AI success with 9/10 pregnancies occurring when follicular size ranged between 33.1–37.4 mm, no pregnancy when it was smaller, and only one when larger. A number of ass species face the risk of extinction. Knowledge gained in this study on the Abyssinian donkey can be customised and transferred to its closely related endangered species and breeds.

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Effect of Post‐Thaw Addition of Seminal Plasma on Motility, Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Cited by 15 publications

References 35 publications

Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

Evaluation of hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability

New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models

Follicular size predicts success in artificial insemination with frozen-thawed sperm in donkeys

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