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.

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

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.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7–8, grade 1–2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Cryopreservation of embryos has the potential to become a valuable tool for the conservation of endangered donkey breeds. However, there are several factors that can affect cryosurvival of embryos. This study evaluates the effectiveness of the Cryotop method to vitrify donkey embryos and factors affecting the survival of vitrified-warmed embryos. Day 6-8 embryos were measured and morphologically evaluated. Embryos were then vitrified-warmed using the Cryotop technique. After 24 h post-warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). A total of 25 embryos were used, of which 17 were classified as Grade 1 (excellent), 5 as Grade 2 (good) and 3 as Grade 3 (fair). Based on their diameter, embryos were grouped as follows: <= 220 mu m (n = 10), >220-300 mu m (n = 8), and >300 mu m (n = 7). Post-warming survival of vitrified embryos was similar (P> 0.05) to the control fresh embryos, regardless of embryonic diameter, developmental stage, and age of the embryos before vitrification. However, the proportion of embryos that survived vitrification procedures was numerically higher but not significantly different (P > 0.05) for Day 7 embryos (84.6%). The ability of Grade 1 (70.6%) and 2 (80%) embryos to survive vitrification procedures was higher (P = 0.0214) than those of Grade 3 (0%). The proportion of dead cells in Grade 3 embryos (56.5%) was higher (P < 0.01) than that of Grade 1 (3.2%) and 2 (1.5%) embryos. In conclusion, the Cryotop technique seems to be useful for Grade 1 and 2 donkey embryos. It is likely that donkey embryos show similar survival rates after vitrification in Cryotops irrespective of age, diameter and development stage. (C) 2018 Elsevier Inc. All rights reserved.

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.

Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 μl), sperm concentrations (50, 100, 200 × 106 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 200 mM) and plunged into LN2. Conventional freezing was performed in 0.5 ml straws frozen in LN2 vapors. Sperm motility, plasma and acrosome membrane integrities and DNA fragmentation were compared among treatments. The use of straws filled with 100 μl at 100 × 106 sperm/ml with the extender containing 100 mM trehalose resulted in greater values for sperm quality than the other concentrations, volumes and carbohydrates. With vitrification, there were greater values (mean ± SEM; P < 0.05) than freezing for progressive motility (48.2 ± 2.3 compared with 37.3 ± 2.2%), plasma membrane integrity (82.8 ± 1.5 compared with 74.1 ± 1.9%), and intact acrosomes (50.2 ± 1.2 compared with 43.1 ± 1.4%); and less DNA fragmentation (6.4 ± 0.7 compared with 8.2 ± 0.3%). In conclusion, stallion sperm can be vitrified in 0.25 ml straws filled with 100 μl of sperm at 100 x 106 sperm/ml using an extender with 100 mM of trehalose, obtaining better sperm quality after warming than conventional freezing.

The aim of this study was to assess different concentrations of sucrose-based extenders combined with bovine serum albumin (BSA) as an alternative to stallion sperm cryopreservation with permeable cryoprotectants. Semen samples (n = 16) were collected from six stallions. Sperm was cooled, filled in 0.5 mL straws and frozen in nitrogen vapor. Post-thaw sperm kinetic parameters, plasma and acrosome membrane integrity were statistically compared among treatments. In Experiment 1, extenders containing 1% of BSA and different concentrations of sucrose (mmol/L, M): 0, 50, 100, 250, 350 and 450 mM were compared. Use of sucrose [100 mM (S2)] resulted in greater values for most of the sperm kinetic parameters assessed (P < 0.001). There were no differences for plasma membrane integrity, except for when sucrose was used at 50 and 250 mM concentrations, and plasma membrane integrity was less (P < 0.05) when these concentrations were used than with the other sucrose concentrations. In Experiment 2, the selected sucrose extender (S2) was compared to an extender containing glycerol as permeable cryoprotectant. Use of the S2 extender resulted in a lesser proportion of sperm with denuded-acrosomes (P < 0.05) in comparison to use of glycerol and values for several kinetic parameters were also greater (P < 0.05) with use of S2. There were no significant differences for the other parameters assessed in this study. In conclusion, stallion sperm can be frozen in the absence of permeable cryoprotectants, using a combination of sucrose 100 mM with BSA-1% as alternative agents.

This study was conducted to evaluate the effect of amino acid addition to semen on post-thaw quality of donkey spermatozoa. Eighteen ejaculates were pooled and divided into aliquots which were cryopreserved in Gent A® containing 1% ethylene glycol (Gent-EG) and supplemented with 0 (as control), 20, 40, or 60 mM of glutamine, proline, or taurine. The greatest concentration (60 mM) of glutamine and taurine resulted in greater (P < 0.001) post-thaw sperm motility. Amino acid supplementation did not improve (P > 0.05) sperm morphology and membrane plasma integrity compared with the control samples. Whereas, improvement (P < 0.05) of acrosome integrity was observed with use of 60 mM glutamine. After thawing, there were no differences (P > 0.05) in the sperm DNA fragmentation index (sDFI) among treatments. The 60 mM glutamine and 40 mM taurine treatments, however, resulted in a reduction (P < 0.05) in sDFI values in the first 6 h of semen incubation, compared with the control samples. At 24 h, the sDFI values were less (P < 0.05) in all supplemented as compared with control samples, except for the 20 mM proline treatment group. In conclusion, supplementation of the Gent-EG extender with glutamine or taurine at 60 mM improved post-thaw donkey sperm quality. The addition of proline to the freezing extender, however, did not provide any significant enhancement in sperm quality, compared with the control group.

Female mules are considered as infertile; however, they could be used as recipients in interspecific embryo transfer. This study reports for the first time how it is possible to obtain the birth of a live Andalusian donkey foal after transfer a donkey embryo to a non-cycling mule. Two non-cycling mules were used as recipients, oestradiol benzoate was administered when donors showed oestrus and long-acting progesterone after ovulation. The mules also received long-acting progesterone every 7 days until 120 days of gestation. One embryo was collected from the two donor jennies and transferred to one of the mules after 5 days of progesterone treatment. Pregnancy was established and maintained after embryo transfer. The pregnant mule carried to term and delivered a live donkey foal after 375 days of pregnancy. In conclusion, non-cycling mules treated with oestradiol benzoate and long-acting progesterone can be successfully used as recipients of donkey embryos, which open new ways for the conservation of endangered donkey species.

The use of ovulation-inducing agents can improve the effectiveness of embryo transfer by synchronizing ovulation and artificial insemination (AI) or mating. Moreover, the duration of oestrus and the number of AIs or matings can be reduced with these treatments. This preliminary study was conducted to compare the efficacy of two drugs, human Chorionic Gonadotrophin (hCG; Veterin Corion, Divasa Farmavic, Spain) and Deslorelin acetate (Sincrorrelin, Ourofino Saude Animal, Brazil), for induction of ovulation in jennies. Data on embryo recovery rates were recorded from 16 Andalusian jennies aged 3-12 years old. A total of 50 oestrus cycles were used and randomly divided into three groups: (i) no treated as control (n = 26), (ii) treated with 1500 IU hCG i.m. (n = 11), and (iii) treated with 0.75 mg Deslorelin acetate i.m. (n = 13). Donors were treated when they had a follicle larger than 35 mm of diameter and uterine oedema. Ovulation was diagnosed by daily ultrasound observations. Six to nine days after ovulation, donors were flushed three times for embryo recovery and each recovered embryo was measured and evaluated for morphology. Data were assessed by the chi-square test and the Kruskal-Wallis ANOVA. All donors (n = 24) responded well to the administration of both ovulation inducing agents. No differences (P > 0.05) were observed between treatments for the time to ovulation from treatment (hCG: 52.00 ± 4.00 h; Deslorelin acetate: 49.60 ± 4.95 h). The range of response was also similar (P > 0.05) for hCG and Deslorelin acetate (24 h: 9.1% vs. 15.4%; 48 h: 72.7% vs. 46.2%; 72 h: 18.2% vs. 38.5%). None of the variables studied (embryo recovery and mean diameter and age of embryos) were affected (P > 0.05) either by the ovulation treatment or the time to ovulation from treatment, but embryo quality (P = 0.019 for hCG vs. Deslorelin acetate). Nineteen embryos were recovered from 26 flushes (73.08%) in control jennies, of which 17 (89.5%) were classified as Grade 1 and 2 (10.5%) as Grade 2, whereas 8 Grade 1 embryos were collected from 11 flushes (72.73%) in jennies treated with hCG. Nine embryos were recovered from 13 flushes (69.23%) in jennies treated with Deslorelin acetate, of which 4 were classified as Grade 1 (44.4%), 3 as Grade 2 (33.3%), and 2 as Grade 3 (22.2%), showing a significantly lower percentage of Grade 1 embryos (P < 0.05) compared to hCG group. In conclusion, our results suggest that both drugs used in this study are adequate to consistently induce ovulation in jennies, but we noted that embryos recovered in jennies treated with hCG had better quality than those of the Deslorelin acetate ones.

Prostaglandin F2alpha (PGF2a) and its analogues have been routinely used after the embryo flush to induce the onset of oestrus in female donors (mares and jennies) and thus to obtain more embryos in a given season. The aim of this preliminary study was to compare the efficacy of Luprostiol (Prosolvin, Virbac, Spain) and Dinoprost tromethamine (Lutalyse, Pfizer Animal Health, Brazil) for induction of oestrus in jennies. Fourteen Andalusian jennies, 3-12 years of age, were used as embryo donors. Ovarian activity was evaluated daily during oestrus by transrectal ultrasound. When a ≥ 35 mm follicle was detected, donor jennies were naturally mated every other day until ovulation (Day 0). Uterine flushing was done on Days 6 to 9. Each recovered embryo was measured and evaluated for morphology. In a total of 38 collections, donors were randomly treated with either 5.25 mg Luprostiol i.m. (n = 27) or 3.5 mg Dinoprost tromethamine i.m. (n = 11) immediately after the embryo recovery. Data were assessed by the chi-square test and the Kruskal-Wallis ANOVA. All donors responded well to prostaglandins (Luprostiol and Dinoprost tromethamine) given right after embryo collection. There were no significant (P > 0.05) differences between treatments for the interval from prostaglandin to ovulation (9.85 ± 0.72 days vs. 10.18 ± 0.84 days). In addition, the interovulatory interval was similar (16.56 ± 0.63 days vs. 17.27 ± 1.23 days; P > 0.05) between prostaglandins. Furthermore, there were no significant (P > 0.05) differences between these treatments for the embryo recovery rate (average, 74.3%) and embryo quality. Eighteen embryos were recovered from 27 flushes in Prosolvin group (66.7%), of which 14 were classified as Grade 1 (77.8%), 3 as Grade 2 (16.7%), and 1 as Grade 3 (5.6%). Meanwhile, nine embryos were recovered from 11 flushes in Lutalyse group (81.8%), of which 7 were classified as Grade 1 (77.8%), 1 as Grade 2 (11.1%), and 1 as Grade 3 (11.1%). Based in our results, we can conclude that both drugs used in the present study are adequate to consistently induce the onset of oestrus in jennies, with no deleterious effects on embryo recovery rate or embryo quality.