Vitrification in straws conserves motility features better than spheres in donkey sperm.

Sperm vitrification as alternative to conventional freezing is increasing in popularity in many species. It has been achieved by direct exposure of diluted semen to liquid nitrogen in spheres or straws. Both techniques have been successfully developed, but they had not been compared yet in donkeys. The aim of this study was to compare these two methods of vitrification for donkey semen. Ejaculates from six Andalusian donkeys were collected and extended in Gent without glycerol supplemented with sucrose 0.1 M (Molar). Samples were slowly cooled at 5°C. For vitrification, 30 µl suspensions (spheres) were dropped directly into liquid nitrogen (LN2 ) or filled in covered 0.25 ml straws and then plunged into the LN2 (straws). For warming, straws and spheres were directly immersed in 3 ml of INRA-96 at 43°C. Total (TM, %) and progressive motility (PM, %) were objectively evaluated by computer-assisted sperm analysis and plasma membrane integrity (PMI, %) by epifluorescence microscopy. Results showed the straw method resulted in significantly higher values than spheres for: TM (54.7% ± 10.1 vs. 28.6% ± 6.5) and PM (44.2% ± 9.4 vs. 17.7% ± 6.4), but no significant differences were found between straws or spheres for PMI (31.5 ± 10.7 vs. 41.6 ± 14.3) respectively. In conclusion, donkey sperm could be vitrified in straws obtaining better sperm motility parameters after warming in comparison to the sphere method.

Comparison of different sucrose-based extenders for stallion sperm vitrification in straws.

Vitrification of sperm is based on high-speed freezing by direct exposure to liquid nitrogen using non-permeable cryoprotectants, mainly disaccharides; yet, the concentration of cryoprotectants has a species-specific effect on the sperm cell. The aim of this study was to assess different sucrose concentrations for stallion sperm vitrification. Semen samples (n = 9) were collected from three stallions, centrifuged and resuspended to a concentration of 50 × 106  sperm/ml in a base extender (INRA96 + 1% of bovine serum albumin) with three different sucrose concentrations (Molar): 20 mM (S1), 100 mM (S2), or 200 mM (S3). Then, sperm were filled in covered 0.25 ml straws and directly plunged into liquid nitrogen. For warming, 0.25 ml straw was pulled out the covering straw and immersed in 3 ml of INRA96 at 43°C, with gentle pipetting to accelerate the melting. Total (TM, %) and progressive sperm motility (PM, %) were analysed using computer-assisted sperm analysis. Plasma (PMI, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post-warmed sperm parameters were compared between treatments by ANOVA. S2 showed significantly higher values in comparison with S1 and S3 for TM (S2 = 54.7 ± 5.5a ; S1 = 29.1 ± 3.3b ; S3 = 28.6 ± 3.0b ; p < 0.001) and PM (S2 = 31.3 ± 3.8a ; S1 = 18.5 ± 2.6b ; S3 = 17.7 ± 2.9b ; p < 0.01), respectively. No significant differences were found among treatments for PMI (S2 = 70.3 ± 5.2; S1 = 67.4 ± 4.3; S3 = 70.0 ± 3.7) neither for AIS (S2 = 57.1 ± 3.9; S1 = 53.9 ± 4.2; S3 = 57.0 ± 7.9). In conclusion, a concentration of 100 mM sucrose is recommended for stallion sperm vitrification in straws.

Identification of sperm morphometric subpopulations in cooled-stored canine sperm and its relation with sperm DNA integrity.

The aims of this study were to (i) identify different morphometric subpopulations in cooled-stored canine sperm and their patterns of distribution during cool-storage for up to 240 hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer-assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72 hr and thereafter sP3 increased progressively. sDFi increased after 48 hr of cool-storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool-storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.

Effect of different extenders for donkey sperm vitrification in straws.

Aseptic vitrification of semen samples packed in straws has been successfully developed in human but not in donkeys. The aim of this study was to compare the effect of two extenders for donkey sperm vitrification using straws. Ejaculates from four Andalusian donkeys were collected, and samples were extended in INRA-96 (I) or Gent (G) supplemented with sucrose 0.25 M and 1% bovine serum albumin (BSA). Extended samples were cooled for one hour at 5°C. For vitrification, samples were filled in covered 0.25 ml straws and then plunged directly into liquid nitrogen. For warming, straws were immersed in INRA-96 at 43°C. Results showed no significant differences between I and G treatments for TM (34.2% ± 8.7 vs. 30.7% ± 9.6) and PM (26.8% ± 7.3 vs. 24.6% ± 7.9), respectively. In conclusion, donkey sperm could be vitrified in straws either with INRA-96 or with Gent in combination with sucrose and BSA.

First case of sterility associated with sex chromosomal abnormalities in a jenny.

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.

Freezability of Andalusian donkey (Equus asinus) spermatozoa: effect of extenders and permeating cryoprotectants.

The aim of this study was to compare the effect of two semen extenders and four permeating cryoprotectants on post-thaw sperm quality of Andalusian donkeys. First, 32 ejaculates were pooled, split and frozen in either Gent B or INRA 96 with egg yolk and glycerol. Second, 12 pooled semen samples were simultaneously frozen in Gent B (glycerol) or Gent A containing ethylene glycol (EG; 1 or 1.5%) or dimethyl sulfoxide (DMSO; 1.5 or 2%). Finally, nine pooled samples were simultaneously cryopreserved in Gent A containing 1% EG (as control), dimethylformamide (DMFA; 1 or 2.5%) or a combination of 1% EG and 1.5% DMFA. Gent B yielded a higher (P<0.01) post-thaw sperm motility than modified INRA96. EG 1% increased the sperm membrane integrity (P<0.001), whereas DMSO affected sperm motility and membrane integrity (P<0.001). DMFA 2.5% yielded higher (P<0.001) values for sperm motility and membrane integrity. We concluded that Gent B improves in vitro post-thaw sperm quality of donkey spermatozoa, but the replacement of glycerol with 1% EG or 2.5% DMFA increased sperm protection against cryodamage. The use of DMSO for freezing donkey semen was unsuccessful and a toxic effect is suspected. These extenders should be included in the pre-freeze test for each donkey.

Colloid single-layer centrifugation improves post-thaw donkey (Equus asinus) sperm quality and is related to ejaculate freezability.

The aim of this study was to determine whether colloid single-layer centrifugation (SLC) improves post-thaw donkey sperm quality and if this potential enhancement is related to ejaculate freezability. Semen from Andalusian donkeys was frozen following a standard protocol. SLC was performed on frozen-thawed semen and post-thaw sperm parameters were compared with uncentrifuged samples. Sperm quality was estimated by integrating in a single value sperm motility (assessed by computer-assisted sperm analysis), morphology and viability (evaluated under brightfield or fluorescence microscopy). Sperm freezability was calculated as the relationship between sperm quality obtained before freezing and after thawing. Ejaculates were classified into low, medium and high freezability groups using the 25th and 75th percentiles as thresholds. All sperm parameters were significantly (P<0.01) higher in SLC-selected samples in comparison to uncentrifuged frozen-thawed semen and several kinematic parameters were even higher than those obtained in fresh semen. The increment of sperm parameters after SLC selection was correlated with ejaculate freezability, obtaining the highest values after SLC in semen samples with low freezability. We concluded that, based on the sperm-quality parameters evaluated, SLC can be a suitable procedure to improve post-thaw sperm quality of cryopreserved donkey semen, in particular for those ejaculates with low freezability.

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques.

This study compared the efficacy of simple sperm washing (SW), single-layer centrifugation (SLC) and modified swim-up (SU) techniques in the preparation of dog spermatozoa for cooling. Eighteen ejaculates, collected from three dogs (six per dog), were pooled (three ejaculates per pool) and divided into three aliquots: (1) one aliquot was washed and cooled at 5°C for 72h, considered as control (SW-control), (2) the second aliquot was selected by SLC through Androcoll-C and subsequently cooled in the same way as the SW-control samples (SLC-AC) and (3) the last aliquot was selected by a modified SU method with Androcoll-C and cooled as mentioned above (SU-AC). Assessment of sperm motility, sperm morphology, sperm membrane integrity and acrosome integrity were performed on aliquots of fresh semen and chilled-rewarmed samples. Sperm membrane integrity and progressive motility were significantly (PPP>0.05). The recovery rates were not significantly (P>0.05) different between SW-control, SLC-AC and SU-AC samples. Our results confirm that SU-AC may be a successful method for the preparation of dog spermatozoa for cooling.

Provisioning floral resources to attract aphidophagous hoverflies (Diptera: Syrphidae) useful for pest management in central Spain.

Noncrop plant communities present on the boundaries or within crop fields are essential for the maintenance of functional biodiversity, affecting beneficial insect numbers and ecological fitness. Habitat manipulation is an increasingly studied strategy aimed at enhancing natural enemies of agricultural pests by providing feeding and shelter resources. In this study, six plant species selected from preliminary work were tested for their potential attractiveness to four common aphidophagous hoverflies species. Potential attractiveness was evaluated through observation of hoverfly feeding visits to replicated flower plots distributed in a randomized design. The combination of the selected species covered a 2-mo full-bloom period. Sphaerophoria scripta L. and Sphaerophoria rueppellii (Wiedeman) were the dominant hoverflies present throughout the sampling period, whereas Eupeodes corollae (F.) and Episyrphus balteatus (DeGeer) visits were less abundant and appeared only in the early season. Potential attractiveness varied among plant species. Calendula arvensis L. and Coriandrum sativum L. were the most visited species. C. arvensis received a high number of visits throughout a long period, whereas the visits to Co. sativum were concentrated in a short blooming period. These results suggest that habitat management by using these plant species may increase the abundance of hoverflies and could improve the biological control of aphid pests typical of spring-summer crops in open Mediterranean environments.

Hybrid model based on Genetic Algorithms and SVM applied to variable selection within fruit juice classification.

Given the background of the use of Neural Networks in problems of apple juice classification, this paper aim at implementing a newly developed method in the field of machine learning: the Support Vector Machines (SVM). Therefore, a hybrid model that combines genetic algorithms and support vector machines is suggested in such a way that, when using SVM as a fitness function of the Genetic Algorithm (GA), the most representative variables for a specific classification problem can be selected.

Clinical, molecular and biochemical characterization of nine Spanish families with Conradi-Hünermann-Happle syndrome: new insights into X-linked dominant chondrodysplasia punctata with a comprehensive review of the literature.

BACKGROUND: Conradi-Hünermann-Happle syndrome (CDPX2, OMIM 302960) is an inherited X-linked dominant variant of chondrodysplasia punctata which primarily affects the skin, bones and eyes. CDPX2 results from mutations in EBP (emopamil binding protein), and presents with increased levels of sterol precursors 8(9)-cholesterol and 8-dehydrocholesterol. OBJECTIVES: To expand the understanding of CDPX2, clinically, biochemically and genetically. METHODS: We present one of the largest series reported to date, including 13 female patients belonging to nine Spanish families. Patients were studied biochemically using gas chromatography-mass spectrometry, genetically using polymerase chain reaction and in their methylation status using the HUMARA assay. RESULTS: In our cases, there was a clear relationship between abnormal sterol profile and the EBP gene mutation. We describe three novel mutations in the EBP gene. EBP mutations were inherited in three out of nine families and were sporadic in the remaining cases. CONCLUSIONS: No clear genotype-phenotype correlation was found. Patients' biochemical profiles did not reveal a relationship between sterol profiles and severity of disease. A skewed X-chromosome inactivation may explain the clinical phenotype in CDPX2 in some familial cases.

Follicular dynamics, interval to ovulation and fertility after AI in short-term progesterone and PGF2α oestrous synchronization protocol in sheep.

The study was aimed to assess the influence that short-term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7-day P4+PGF2α protocol using a new (n = 20) or a 7-day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short-term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1-2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.

Epidemiology of mechanical ventilation in Argentina. The EpVAr multicenter observational study.

OBJETIVE: To describe mechanical ventilation (MV) practices in Argentina, and to explore factors associated with ICU mortality in this population. DESIGN: A prospective, multicenter, observational study was carried out. SETTING: Intensive Care. PATIENTS: We enrolled patients above 18 years old admitted to any of the participating ICUs requiring invasive MV for at least 12 h since the admission to the healthcare institution, including MV initiation in emergency department, operating room or other hospitals. INTERVENTIONS: None. VARIABLES: All variables were classified into three categories: variables related to demographic and clinical factors before the MV, factors related to the first day on MV, and factors related to events happening during the MV (complications and weaning from MV). Mechanical ventilation weaning and mortality were classified according to WIND. RESULTS: The primary analysis included 950 patients. The main indication for MV was acute respiratory failure (58% of patients). Initial ventilation mode was volume control-continuous mandatory ventilation in 75% of cases. ICU and hospital mortality were 44.6% and 47.9% respectively. The variables identified as independent predictors of mortality in ICU were age (OR 3.48 IC 95% 1.22-11.66; p = 0.028), failure to implement NIV before MV (OR 2.76 IC 95% 1.02-7.10; p = 0.038), diagnosis of sepsis (OR 2.46 IC 95% 1.09-5.47; p = 0.027) and extubation failure (OR 4.50 IC 95% 2.05-9.90; p < 0.001). CONCLUSIONS: The present study allowed us to describe the characteristics and clinical course of the patients who received mechanical ventilation in Argentina, finding as the main result that mortality was higher than that reported in international studies.

Overproduction of cyclo-oxygenase-2 (COX-2) is involved in the resistance to apoptosis in vascular smooth muscle cells from diabetic patients: a link between inflammation and apoptosis.

AIMS/HYPOTHESIS: Inflammation is a common feature in cardiovascular diseases, including diabetes mellitus. In addition to the well-known inflammatory role of cyclo-oxygenase-2 (COX-2), this protein has also been implicated in apoptosis resistance in tumour cells. Vascular smooth muscle cells (VSMC) from diabetic patients are also resistant to apoptosis because of an increased abundance of B cell lymphoma 2 protein (BCL2). In this work, we investigated whether overproduction of COX-2 was involved in the resistance to apoptosis in VSMC from diabetic patients. METHODS: VSMC were obtained from internal mammary arteries from patients who had undergone coronary artery bypass graft surgery. Apoptosis was measured by DNA fragmentation, BCL2 degradation and cytochrome c release. RESULTS: Apoptosis induced by C-reactive protein in cells from non-diabetic patients was mediated by COX-2. VSMC from diabetic patients showed higher basal levels of COX-2 compared with those from non-diabetic patients. Transfection of VSMC from non-diabetic patients with a plasmid containing COX-2 (also known as PTGS2) increased basal production of COX-2 and BCL2 and mimicked the resistance to apoptosis that occurs in diabetic patients. We also found a significant correlation (R = 0.846, p = 0.016) between COX-2 and BCL2 production in arterial rings from diabetic patients measured by confocal microscopy. However, inhibition of COX-2 production by small interfering RNA proved unable to reverse BCL2 production in diabetic VSMC. CONCLUSIONS/INTERPRETATION: These results suggest a link between inflammation (COX-2) and apoptosis resistance (BCL2) in the arteries of diabetic patients. This relationship is not causative and the common production of these two proteins may be co-regulated by shared regulatory elements in diabetes.

Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome.

Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein-protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigmatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs.

Antimicrobial activity of silver-carbon nanoparticles on the bacterial flora of bull semen.

The spermicidal effects of silver nanoparticles (AgNPs) hinder its application in the field of artificial insemination. In this study, silver-carbon NPs (Ag@C NPs) was synthesized and applied as an alternative antibiotic agent for bull semen extender. Ag@C NPs were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic absorption flame spectroscopy, transmission electron microscope (TEM), and high-resolution TEM (HR-TEM). Data analysis revealed the successful synthesis of Ag@C NPs with a particle size of 1-5 nm (average particle size of 2.5 nm) embedded into carbon. The antimicrobial activity of Ag@C NPs was tested against bacteriospermia of fresh semen collected from five fertile bulls (three ejaculates/bull). Escherichia coli (E. Coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were isolated from fresh semen samples and identified by culture, staining, and conventional biochemical tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag@C NPs against bacteriospermia was determined at 5 and 37 °C. Ag@C NPs showed efficient antimicrobial activity (MIC: 3.125-12.5 µg/mL) against the tested strains and strong bactericidal effect on S. aureus, and P. aeruginosa (MBC: 3.125 µg/mL), with no detrimental effect (P ˃ 0.05) on the percentage of sperm motility (70.71 ± 4.82; 74.65 ± 4.46), plasma membrane integrity (68.39 ± 4.31; 72.38 ± 4.91), acrosome integrity (88.40 ± 13.21; 86.77 ± 14.23), and normal sperm morphology (86.85 ± 7.43; 87.82 ± 8.15) at concentrations of 15 and 30 µg/mL, respectively, after a cold storage of 48 h. However, Ag@C NPs showed a detrimental effect on sperm parameters in a dose dependent manner at concentrations ≥60 µg/mL. Ag@C NPs showed no adverse effect on the sperm's ultrastructure with limited sperm internalization at MIC. In conclusion, Ag@C NPs could be used as an alternative antibiotic agent for bull semen extender without a significant cytotoxic effect on the sperm during cold storage. However, further investigations for their effects on embryo production and female genitalia are still required.

First pregnancies in jennies with vitrified donkey semen using a new warming method.

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 106 sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ±â€¯13.2%) and progressive sperm motility (41.4 ±â€¯11.4%). No differences in PMN concentration (×103 PMN/ml) were found between vitrified (276.8 ±â€¯171.6) or frozen (309.7 ±â€¯250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies.

One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos.

The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7-8, grade 1-2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.

Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer).

The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.