Vitrification of donkey sperm using straws as an alternative to conventional slow freezing.

Cryoprotectant-free vitrification of donkey sperm using 0.25 ml straws has been recently developed, but the obtained results have not been directly compared to conventional slow freezing yet. The aim of this study was to compare sperm quality parameters after cryopreservation using both methods. Semen samples were collected from three Andalusian Donkeys. Semen was centrifuged and pellets resuspended with an extender with glycerol for conventional freezing or the same extender without glycerol, but with sucrose 0.1 mol/L for vitrification. Conventional freezing was performed in nitrogen vapours and thawed in a water bath (30s/37°C). Vitrification was performed in covered 0.25 ml straws plunged directly into liquid nitrogen and warmed in 3 ml of a milk-based extender at 43°C. Total (TM, %) and progressive motility (PM, %) were evaluated by computer-assisted sperm analysis, and plasma membrane (PMI, %) and acrosome (AIS, %) integrities by epifluorescence microscopy. No differences (p > .05) were found between slow freezing and vitrification methods for any of the parameters assessed: TM (58.2 ± 16.1% vs. 52.7 ± 15.6%), PM (44.7 ± 18.2% vs. 44.3 ± 15.0%), PMI (55.4 ± 9.0% vs. 49.2 ± 11.2%) and AIS (38.4 ± 19.6% vs. 45.0 ± 11.0%), respectively. In conclusion, donkey sperm vitrification in straws presented similar sperm quality after thawing in comparison to conventional freezing. Therefore, it could be considered as an alternative to slow freezing regarding the sperm parameters assessed.

DNA fragmentation of equine cumulus cells from Cumulus-Oocyte complexes submitted to vitrification and its relationship to the developmental competence of the oocyte.

The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs. The percentage of CC-DF was not significantly different (p > .05) between groups of COCs able to reach metaphase II (MII > 0) and those in which oocyte maturation was not achieved (MII = 0). In conclusion, vitrification has a deleterious effect on the DNA fragmentation of equine cumulus cells; however, this parameter cannot be used as a predictor for IVM success after COCs vitrification.

Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled-stored stallion sperm.

Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24 hr of storage. The coefficient of determination (R2 ) was calculated after fine-tuning linear (LIN), exponential (EXP) and second order polynomial (POL) models. R2 was significantly higher (p < .001) for POL than for LIN and EXP. The rate of DNA degradation was calculated using the slopes of POL equations. After assessing the rate of change of the POL functions, significant differences between the acceleration of DNA fragmentation were found (p < .01) among seasons, being higher for winter and summer than spring and autumn. In conclusion, DNA analysis of stallion sperm fits better to a second order polynomial mathematical model, being spring the best season to collect and process cooled stallion semen in order to maintain the DNA integrity of the stallion sperm.

Relationship between DNA fragmentation of equine granulosa cells and oocyte meiotic competence after in vitro maturation.

The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus-oocytes complexes (n = 50) were recovered from slaughterhouse-derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.

Low-density lipoproteins and milk serum proteins improve the quality of stallion sperm after vitrification in straws.

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25-ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post-warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non-permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.

Effect of permeable cryoprotectant-free vitrification on DNA fragmentation of equine oocyte-cumulus cells.

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post-mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus-oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO2 ) before (control group) or after a permeable cryoprotectant-free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at -80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant-free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.

Subcutaneous Administration of Medications and Fluids by Nonprofessional Caregivers at Home.

Background: Patients requiring home-based palliative care have advanced complex illnesses with functional limitations and decline. This retrospective study reviewed caregiver administration of subcutaneous (SQ) medications and fluids when symptom control could not be achieved using the oral route. Methods: Medical records from September 1, 2017 to February 28, 2018 were reviewed for 272 consecutive patients who received SQ administration of medications or fluids at a home-based palliative care program. We analyzed the clinical characteristics of patients and caregivers, medications administered, and catheter outcomes. Results: Patients' median age was 74 years, and 163 (60%) were women. The most common cancer diagnoses were stomach 26 (12%), lung 22 (10%), and colorectal 20 (9%). Dementia 24 (44%), cerebrovascular disease 9 (16%), and congestive heart failure 7 (13%) were the most frequent nonmalignant diseases. Poor symptom control 162 (60%) and impaired oral intake 107 (39%) were the most common indications for an SQ route of administration. Nonprofessional caregivers trained by a nurse administered medications to 218 patients (80%). During interventions, the patients received a mean of 4 medications (±2 standard deviation). A total of 903 catheters were inserted, 15/732 (2%) catheters handled by nonprofessional caregivers caused a local infection, compared with 3/171 (1.8%) of catheters handled by nurses. Hydromorphone was the most common opioid used (57%), followed by morphine (35%). The median length of stay in the program was 24 days (interquartile range: 11-60). Conclusions: SQ administration of medications and fluids by nonprofessional caregivers trained by health care professionals is feasible and promising, but additional testing is needed.

Bicarbonate-Triggered In Vitro Capacitation of Boar Spermatozoa Conveys an Increased Relative Abundance of the Canonical Transient Receptor Potential Cation (TRPC) Channels 3, 4, 6 and 7 and of CatSper-γ Subunit mRNA Transcripts.

Sperm capacitation is a stepwise complex biochemical process towards fertilization. It includes a crucial early calcium (Ca2+) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts changes of the CatSper ß, γ and δ subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control-fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3, 2.25 mM CaCl2, 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 °C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relative abundance of mRNA transcripts of almost all studied Ca2+ channels, except CatSper-δ and TRPC1 (significantly reduced). These findings open new avenues of research to identify the specific role of each channel in boar sperm capacitation and elucidate the physiological meaning of the changes on sperm mRNA cargo.

Hormonal Management for the Induction of Luteolysis and Ovulation in Andalusian Jennies: Effect on Reproductive Performance, Embryo Quality and Recovery Rate.

Two prostanglandins (luprostiol, LUP, and dinoprost, DIN) and two ovulation-inducing agents (human Chorionic Gonadotropin, hCG, and deslorelin, DES) were evaluated for luteolysis and estrus induction, and for ovulation induction, respectively, in embryo donor jennies. Twenty-six fertile Andalusian jennies were used. In Experiment 1, jennies (n = 112 cycles) were randomly treated with either LUP or DIN after embryo flushing. In Experiment 2, donors (n = 84 cycles) were randomly treated with either hCG or DES to induce ovulation. No differences were found between prostaglandins for all variables studied (prostaglandin-ovulation interval (POI), interovulatory interval (IOI), embryo recovery rate (ERR), positive flushing rate (PFR) and embryo grade (EG)). The ovulation rate was similar for hCG and DES (60.9% vs. 78.7%). However, the interval to ovulation (ITO) was affected (62.61 ± 7.20 vs. 48.79 ± 2.69 h). None of the other variables studied (ERR, PFR and EG) were affected (p > 0.05), except for embryo quality (p = 0.009). In short, both prostaglandins evaluated are adequate to induce luteolysis and estrus. Both ovulation-inducing agents hastened ovulation, but DES seems to be more effective than hCG. Follicular diameter affected the interval from treatment to ovulation, and high uterine edema was related to low embryo quality.

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 µL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes.

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 µg/mL; P1 and 2.5 µg/mL; P2) combined with 50 µg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

Vitrification of Large Volumes of Stallion Sperm in Comparison With Spheres and Conventional Freezing: Effect of Warming Procedures and Sperm Selection.

Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 µL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN2) and conventional freezing using 0.5 mL straws frozen in LN2 vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Significant higher values (P < .001) were obtained after vitrification using spheres in comparison with conventional freezing and vitrification in straws for TM (54.46 ± 3.2 vs. 36.47 ± 3.2 vs. 2.50 ± 1.2, %), PM (38.63 ± 3.4 vs. 15.11 ± 2.0 vs. 1.9 ± 0.9, %), IMS (65.40 ± 2.8 vs. 50.50 ± 2.8 vs. 21.63 ± 2.1, %), and AIS (48.89 ± 2.8 vs. 15.46 ± 1.7 vs. 4.69 ± 0.9, %). No differences were found between warming procedures. Single-layer centrifugation after warming at 42°C/20 seconds obtained higher values (P < .05) than unselected samples for TM (32.52 ± 5.8%), PM (14.22 ± 2.8%), IMS (60.01 ± 3.2%), and AIS (44.5 ± 2.2%), whereas selection after 60°C/15 seconds increased TM (23.11 ± 4.3%) and IMS (67.11 ± 3.9%). In conclusion, vitrification in spheres obtained better sperm quality than conventional freezing and vitrification in straws. Warming procedures did not affect the sperm quality but SLC could be a strategy to enhance the quality of the samples after sperm vitrification using 0.5 mL straws.

Comparison of sperm selection techniques in donkeys: motile subpopulations from a practical point of view.

The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

Evaluation of DNA Damage of Mare Granulosa Cells Before and After Cryopreservation Using a Chromatin Dispersion Test.

DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liquid nitrogen (LN2) and then stored at -80°C; P2 = LN2/-80°C adding cryoprotectants (7.5% ethylene glycol + 7.5% dimethyl sulfoxide); P3 = -80°C; P4 = -80°C + cryoprotectants. Granulosa cell samples were processed with the prototype D3-Ovoselect, Halotech DNA, Spain), and DNA was visualized under fluorescence microscopy. High, low, and total DNA fragmentation percentages were compared among treatments by analysis of variance. Results were expressed as mean ± standard error. No significant differences (P > .05) were found among treatments and the control group. Therefore, the four conservation protocols could be considered equally efficient for DNA preservation of granulosa cells from mare oocytes. In conclusion, cryopreservation of granulosa cells in any of the four protocols used adequately preserved the DNA for further analysis.