Augusto Carluccio

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa.

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 x 10(6) sperm/mL limited the devices ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 x 10(6) sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 x 10(6) sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.

Effect of dietary antioxidant supplementation on fresh semen quality in stallion.

In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after supplementation. Dietary supplementation with experimental antioxidants resulted in a significant increase in average path velocity (121.9 ± 3.1 μm/sec in TG vs 118.9 ± 4.3 μm/sec in CG), straightness (86.2 ± 2.4 % vs 82.6 ± 3.9 % in TG and CG respectively), viability (75.6 ± 10.2 % in TG vs 72.3 ± 6.9 % in CG) and total seminal plasma antioxidants levels (2.7 ± 0.5 mmol/l vs 1.9 ± 0.4 mmol/l in TG and CG respectively) while progressive motility 69.7 ± 11 % vs 62.2 ± 9.3 % in TG and CG stallions respectively) and abnormal sperm morphology (8.2±1.5 % in TG vs 14.4±4 % in CG) significantly improved in treated stallions after 60 d of supplementation. In contrast with previously reported in other species, a negative effect of antioxidant supplementation on semen concentration was recorded in the TG. A positive correlation between progressive motility and total antioxidants in seminal plasma in both treated and control stallions suggested that motility is affected by oxidative-antioxidative status, and that dietary antioxidant supplementation could increase the ability of spermatozoa to contrast reactive oxygen species or the ability of seminal plasma to reduce the oxidative stress. The improvement of semen parameters after antioxidant supplementation was not linear, and after 30 d (or 60 d for some parameters), a further increase was not noted. This evidence suggested that in our standard conditions, dietary intake of these antioxidants could be slightly under the dietary requirement and further evaluation of the actual nutrition requirements of organic selenium, zinc, and vitamin E in the stallion are needed.

Oxytocin, vasopressin, prostaglandin F2α, luteinizing hormone, testosterone, estrone sulfate, and cortisol plasma concentrations after sexual stimulation in stallions

This experiment was designed to determine the effects of sexual stimulation on plasma concentrations of oxytocin (OT), vasopressin (VP), 15-ketodihydro-PGF(2alpha) (PG-metabolite), luteinizing hormone (LH), testosterone (T), estrone sulfate (ES), and cortisol (C) in stallions. Semen samples were collected from 14 light horse stallions (Equus caballus) of proven fertility using a Missouri model artificial vagina. Blood samples were collected at 15, 12, 9, 6, and 3 min before estrous mare exposure, at erection, at ejaculation, and at 3, 6, and 9 min after ejaculation. Afterwards, blood sampling was performed every 10 min for the following 60 min. Sexual activity determined an increase in plasma concentrations of OT, VP, C, PG-metabolite, and ES and caused no changes in LH and T concentrations. The finding of a negative correlation between C and VP at erection, and between C and T before erection and at the time of erection, could be explained by a possible inhibitory role exerted by C in the mechanism of sexual arousal described for men.

Efficiency of different extenders on cooled semen collected during long and short day length seasons in Martina Franca donkey.

Artificial insemination with cooled semen is routine in equids because of its good fertility rates and relatively low costs. In several donkey breeds, especially in restricted populations, the use of cooled semen could be seen as the best way of improving reproductive performance and avoiding excessive inbreeding. Furthermore, most jennies have ovulatory estrous throughout the year, and thus, cooled semen could also be used during short day length season. The aims of this study were to evaluate the effects of different extenders on sperm quality during cooling in the Martina Franca breed, and to verify the preservation of cooled semen collected during long day length (May-June) and short day length (November-December) seasons. Three ejaculates were collected at 10-day intervals from each of six jackasses during both May-June and again in November-December time periods. Each ejaculate was cooled in INRA96 or E-Z Mixin at a low cooling rate and evaluated daily over a 120-h preservation time. The results showed a significant extender influence on preservation time in both periods. Semen diluted with INRA96 maintained a progressive motility of 36% and a straightness of 89% at 120h, whereas semen extended with E-Z Mixin had a mean progressive motility of 32% and a straightness of 81% at 48h during the May-June period. Despite having the same initial characteristics, semen collected during the short day length season had a higher rate of decline in semen quality during storage at 5 degrees C with E-Z Mixin.

Blood analysis in newborn donkeys: hematology, biochemistry, and blood gases analysis

The knowledge of reference ranges for hematologic, biochemical, and blood gas parameters in the different species and the influence of breed and age on them is a fundamental tool for the clinician. For this reason, the aim of this study was to evaluate the age-related changes of hematologic and biochemical parameters in Martina Franca donkey foals during the first 3 weeks of life and of blood gases during the first 24 hours of age. Fifteen healthy donkey foals were enrolled; blood samples were collected from each foal at 10 minutes after birth, 1 hour after the first and second suckles, 12 and 24 hours after birth, daily from Day 2 to 7, and at Days 10, 14, and 21 of life. Erythrocytes, leukocytes, and platelets counts were assessed; also metabolic (alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatinphospokinase, lactate dehydrogenase, alkaline phosphatase, glucose, blood urea nitrogen, creatinine, total proteins, albumins, cholesterol, and total bilirubin) and electrolytic parameters (Ca, P, Mg, Na, K, and Cl) were evaluated. Finally, blood gases and metabolic parameters (pH, pCO2, pO2, sO2, TCO2, HCO3, lactate, and base excess) on venous blood were assessed with a portable analyzer. A statistical analysis to evaluate the influence of age and sex was performed. Several differences were found between sampling times, demonstrating that age influences these parameters. Moreover differences were found compared with data reported in literature for donkey foals of another species, horse foals, and adult donkeys. Although a great interindividual variation for some parameters exists, this study demonstrated that interval references should be addressed not only to different species, but also to specific breeds and to the neonatal period.

Kinematic study on the effect of pH on bull sperm function

Since the mammalian spermatozoa became capable of motion, during the epididymal transit, the spermatozoon swims in a liquid medium and it is completely dependent on the environmental conditions. Some reports have suggested an influence of pH on sperm kinetic characteristics, but no study has objectively described how motility changes in a different environmental pH. In this study, we evaluated the effect of different environmental pHs (5.5, 6, 6.5, 7, 7.5, 8, and 8.5) on kinetic parameters, sperm viability, mitochondrial activity, and sperm morphology of bull semen immediately and 1h after dilution. The results showed higher values for sperm motility characteristics, viability, and mitochondrial activity at pH 7 and 7.5. Values of pH lower than 6.5 and higher than 8 resulted in suboptimal motility, with a decrease in most parameters. At pH 8 and 8.5, a discrepancy between viability and total and progressive motility was found, with a significant amount of spermatozoa that were live but immotile. This reduction seemed related to a decrease in mitochondrial activity, possibly due to the increase in pH. The flow cytometric evaluation of sperm viability assessed by calcein AM was very consistent with the amount of spermatozoa with membrane integrity, evaluated in fluorescence by propidium iodide/SYBR-14 stain. Thus, the calcein AM stain could be used as viability stain instead the classic propidium iodide/SYBR-14 stain because this could allow the addiction of other functional stains without a overlapping of the fluorescent signal in the flow cytometer.

A comparative stereological study of the term placenta in the donkey, pony and Thoroughbred.

The aim of the study was to compare horse and donkey placentae using stereological techniques. Term placentae were collected at spontaneous foaling from seven Thoroughbred mares, seven pony mares, and six jenny donkeys. Maternal and foal weights were recorded and the mass, volume, and gross area of each allantochorion was also recorded. Ten random biopsies were recovered and processed for light microscopy from which the surface density of the microcotyledons (S(v)) and the total microscopic area of fetomaternal contact were calculated stereologically. Gestation length was longer in the donkeys than the other two groups (median values: 371 vs. 327 and 341 days, P < 0.05). There were significant correlations between foal birthweight and gross area (rho = 0.89; n = 20; P < 0.05), mass (rho = 0.84; n = 20; P < 0.05) and volume (rho = 0.89; n = 20; P < 0.05) of the allantochorion. S(v) was higher in the donkey placenta than the other groups (median values: 0.05 vs. 0.03 and 0.04 microm(-1), P < 0.05) although placental efficiency was lower in the donkeys (median values: 0.87 vs. 1.33 and 1.32 kg/m2, P < 0.01). The results of the study confirmed that, although strong morphological similarities exist between the allantochorion of the horse and donkey, that of the donkey develops more complex microcotyledons, as judged stereologically, and exhibits a lower placental efficiency. These differences may be related to maternal genotype and/or the longer gestation length shown by the donkey compared to the horse, but a negative correlation (rho = -0.92, P < 0.01) was also found between age and placental efficiency in donkeys.

Electrolytes changes in mammary secretions before foaling in jennies

No knowledge regarding the peripartum changes in mammary secretions in the jenny are presently available in literature. In the mare, instead, several studies report the role of these changes as indicators of foetal readiness for birth and impending parturition. This experiment was designed to determine calcium, sodium, potassium concentrations, and the value of sodium/potassium ratio in mammary fluids during prepartum in the jenny. Samples were daily collected by hand milking, after mammary gland size increased noticeably, from 17 Martina Franca jennies. Prepartum mammary secretions were analysed every other day between day 10 and day 2 antepartum, and then once a day from the day before to the day of parturition. Calcium concentration showed a significant increase between day 10 and day 6 antepartum and then between day 6 and days 4 and 2. Afterwards, another statistical significant increase was observed at parturition. Sodium concentration significantly decreased from day 10 to day 2 prepartum. Potassium concentration significantly increased between day 10 and day 8 before parturition, then showed a further increase at day 4, followed by none significant changes until foaling. All jennies showed a reversal in sodium/potassium ratio between 2 days antepartum and the day before. In conclusion, the evaluation of mammary fluid calcium concentrations and the reversal of sodium/potassium ratio could be used as good indicators of foetal maturity in the jenny. As far as the prediction of parturition is concerned, the reversal of sodium/potassium ratio is the best parameter, since it was detected 48-24 h before parturition in all considered animals.

Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.

Effect of sperm concentration on characteristics of frozen-thawed semen in donkeys

In this study, the effect of donkey sperm concentration in the straw during cryopreservation on the quality of thawed semen was evaluated. Samples from seven adult Martina Franca jackasses were collected three times using a Missouri artificial vagina. After estimation of volume and concentration, raw semen was evaluated for motility using a computer-assisted sperm analyzer (CASA); viability and acrosome integrity were also determined. Fresh semen was then centrifuged and re-suspended at five different concentrations (100, 250, 500, 750, and 1000×10(6)sperm/ml) with a commercial extender, packaged in 0.5ml straws, and frozen. After thawing, motility parameters, viability, and acrosome integrity were analyzed. The analysis of the data showed similar parameters of fresh semen compared with those of centrifuged and cooled samples. The sperm concentration in the straw affected the semen parameters analyzed after thawing, as suggested by evidence that when the concentration increased, the quality of the post-thawed semen decreased. Furthermore, the differences in total and progressive motility among samples at different concentrations are due to the immobilization of spermatozoa, as suggested by the finding that the percentage of static spermatozoa increased when the concentration increased. The reason for the impairment of semen quality when the sperm concentration increased was discussed. A great variability in cryo-resistance was found between jackasses but not within the same male, suggesting the presence of donkey males with semen that has acceptable and unacceptable freezing qualities.