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BACKGROUND: Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. METHODS: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. RESULTS: Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45 min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl-alcohol = 0.76 ± 0.01 vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). CONCLUSION: These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

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Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Assuntos

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Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Assuntos

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The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species.

Assuntos

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Due to the present pandemic situation and the many animal species that are epidemiologically involved, there has been a surge of renewed interest in investigating the coronavirus (CoV) population circulating in wildlife, especially bats and rodents, which are potential reservoirs of new human pathogens. In Argentina, information about the viruses present in these mammals is very limited. To investigate the presence of coronaviruses in this country, we obtained 457 samples from hematophagous, insectivorous, and frugivorous bats and rodents from two regions of Argentina. We report here the detection of alphacoronavirus sequences in three groups of bats as well as in rodents. Phylogenetic analysis showed the closest relationships to alphacoronaviruses from Brazil.

Assuntos

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C11-BODIPY581/591 is a fluorescent probe that has been successfully used to evaluate lipid peroxidation in different species, but it has not been completely studied in the dog. Thus, the aim of the present study was to assess lipid peroxidation of dog spermatozoa using C11-BODIPY581/591 and compare different positive controls of the technique. Twenty-four ejaculates were collected from 8 adult male dogs. Routine seminal characteristics were evaluated in raw semen. Lipid peroxidation evaluation was performed as described in other species. Samples were divided in three aliquots, exposed to UV radiation, incubated with hydrogen peroxide or left without treatment (control). Lipid peroxidation was significantly greater only in UV-exposed samples than in the control ones (91 ± 6% vs. 8.3 ± 3.5%, p Ë‚ .01). In conclusion, C11-BODIPY581/591 is useful to evaluate lipid peroxidation of dog spermatozoa and UV radiation is a good promoter of membrane oxidation, so irradiated samples can be used as a positive control of this technique.

Assuntos

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Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal-Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.

Assuntos

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The aim of the present study was to evaluate the application of a GnRH-PGF2α based synchronization and superstimulation protocol for fixed-time natural mating in llama embryo donors. All females (n = 8) received 8 µg IM of GnRH analog (GnRHa; buserelin) on day 0, regardless of follicular status. After eight days, another GnRHa dose was administered followed by 250 µg IM PGF2α (cloprostenol). A dose of 1000 IU IM of equine chorionic gonadotrophin (eCG) was applied on day 12 and a new dose of PGF2α was administered on day 13. All embryo donors were mated with a male of proven fertility followed by a GnRHa dose on day 18. 24 h later, mating was repeated with a different male. Transcervical uterine flushing for embryo recovery was carried out on all females on day 26. Recipient females received one dose of GnRHa (day 0) two days after the first mating of embryo donor females. A 75% (6/8) of embryo donors responded to the superstimulation treatment with a range of 2 to 5 corpus luteums (CLs) on embryo recovery day. A total of 24 CLs were registered, with a mean of 4 ± 0.9 CLs per female. Embryo recovery rate was 66.7% (16/24), with a range of 0 to 4 embryos and a mean of 2.7 ± 1.5 embryos per female. Regarding quality of the recovered embryos, 56.2% were grade I, 6.2% were grade II and 37.5% were grade V (untransferable; arrested morulae). Grade I and II embryos (n = 10) were transcervically transferred into recipient females (n = 10) six days after inducing their ovulation. At 24 days after embryo transfer (ET), a 50% pregnancy rate was registered. In conclusion, a group of llama embryo donors can be synchronized and superstimulated using a fixed-time mating protocol based on GnRHa, PGF2α, and eCG without the necessity of using ultrasonography in the field.

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The aim of this study was to assess the uterine blood flow (UBF) and corpus luteum blood flow (CLBF) in llamas 8 days post-mating, using color-Doppler ultrasonography (CDU), to determine the possible relationship between vascularization and the presence of an embryo. Adult females (n = 25) were used to monitor ovarian dynamics by palpation and transrectal ultrasonography until detection of a ≥6 mm growing follicle. Females were randomly assigned to one of two groups: Group I (n = 19), were mated and ovulation was induced by a single dose of buserelin (GnRH analog) that same day (Day 0); and Group II (n = 6), only ovulation was induced (control). On Day 8, UBF and CLBF were evaluated transrectally in both groups. The color-flow images obtained were analyzed with Image J1.52a software to determine the vascularization area and the percentage of corpus luteum with blood flow emission (CLBF%) together with the percentage for each uterine horn (UBF%). Statistical analysis was performed using an ANOVA test. In Group I, uterine flushing was performed to obtain the embryos, thus dividing the females into Group I+ (n = 10), when an embryo was recovered and Group I- (n = 9), when no embryo was recovered. Embryo recovery rate was 52.63% (10/19). In Group I+, UBF% was significantly higher compared to Group I- and Group II (P <0.05). UBF appears to be a good predictor for embryo presence, with an area under the curve (AUC) of 0.9 and an optimal cut-off value of 9.37% (with a sensitivity of 90% and specificity of 88.9%). The CLBF% did not differ between groups (P > 0.05). In conclusion, it is possible to detect a local increase of UBF in the presence of an embryo on day 8 post-mating in llamas. This could be useful to achieve an early pregnancy diagnosis or to decide whether to carry out the uterine flushing in a llama embryo transfer program.