Domenico Robbe

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.

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.

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.

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.

The effect of the chamber on kinetic results in cryopreserved bull spermatozoa

In this study, the effect of the chamber used for the automated analysis of sperm motility by a computer‐assisted semen analysis system on sperm kinematics was evaluated, and the cause of this effect was also verified. Twenty‐three bull semen batches were thawed, and semen was diluted, aliquoted and analysed with six different chambers, (three capillary‐loaded chambers and three droplet (DR)‐loaded chambers). For each chamber type, each sample was analysed in quadruplicate, and the reliability of the analysis was tested using an intraclass correlation coefficient (ICC). Furthermore, sperm membrane integrity (MI) was evaluated, for each sample and chamber, in 12 randomly selected central and 12 edge fields. The ICC analysis showed that some parameters could have a significant variability related to the chamber. High stability of results was detected in Leja 4‐chamber slide. Furthermore, as previously reported in other studies, capillary‐loaded chambers seemed to affect the total and progressive motility and sperm velocities (average path velocity, straight line velocity and curvilinear velocity). These findings were corroborated by the evaluation of sperm MI that was significantly higher in the DR‐loaded chambers. This study confirms that the chamber used for the objective kinetic evaluation of bull‐thawed spermatozoa significantly affects the result. These differences could be present also in other species, even if the specific effect on the sperm kinematics should be verified. The Makler chamber seemed to give reliable results with negligible effects on sperm kinematics.

Effect of the season on some aspects of the estrous cycle in Martina Franca donkey

The Martina Franca (MF) donkey breed, with 48 jackasses and 515 jennies, is considered an endangered breed according to the data from the Monitoring Institute for Rare Breeds and Seeds in Europe. The knowledge of the estrous cycle characteristics has a great impact for assisted reproduction, especially in endangered species. In this study, the estrous cycle characteristics were investigated in 12 MF jennies throughout the year. Estrous cycle, estrous and diestrous lengths, follicular growth and ovulation, and estradiol-17β (E2) and progesterone (P4) plasma concentrations were monitored in MF jennies and compared in different seasons. In all jennies (100%) estrous cycle was detected during the whole year, with no differences in the estrous cycle length among seasons. However, a significant increase of estrous length in spring and summer compared with autumn and winter was found. Diestrus was shorter in summer than in the other seasons. Estrous behavior was always shown and characterized by rhythmic eversion of the vulvar labia (winking) with exhibition of the clitoris, urination, male receptivity and clapping, with sialorrhoea, neck and head extension, and back ears. Estrus was characterized by the ovulation of a larger follicle in spring and summer than in autumn and winter. The pattern of E2 and P4 plasma concentrations during the estrous cycle were similar to that reported for the mare, but without differences among the four seasons, so that a negligible effect of environmental conditions on ovarian E2 and P4 secretion was hypothesized, despite the larger diameter of the ovulating follicle in spring and summer.

Peripartal plasma concentrations of 15-ketodihydro-PGF2α, cortisol, progesterone and 17-β-estradiol in Martina Franca jennies.

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF(2α) (PGFM), cortisol (C), progesterone (P4), and 17β-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at -2.5 days was significantly different compared to samples obtained between -10 and -4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between -3 and -1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates.

BackgroundDensity gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation, was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars, but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.ResultsSingle layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.ConclusionsThese data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.

Influence of season on testicular morphometry and semen characteristics in Martina Franca jackasses

As other European donkey breeds, Martina Franca could be considered an endangered breed because of the population number (48 jackasses and 515 jennies in 2011). To increase donkey population and breed biodiversity preservation, several research projects on donkey reproduction have been performed; however reproductive seasonality has been only partially investigated in jackasses. For this reason the aim of this study was to investigate the effect of seasons and of long (spring to summer; SS) and short (autumn to winter; AW) day length periods, on reproductive physiology of Martina Franca jackasses, in particular on: (1) testicular morphometric characteristics, (2) behavior, through the evaluation of the reaction time, and finally, (3) semen characteristics. Seven adult and reproductively mature Martina Franca jackasses were enrolled. For each jackass, a morphometric evaluation of both testes was performed once for every season, before the first seasonal collection. Semen was collected, by artificial vagina, weekly for a whole year (52 collections per donkey); at each collection the reaction time was recorded and a complete semen evaluation was performed immediately after collection. No differences in testicular measures were observed neither between left and right testis nor during the four seasons. A lower reaction time was observed in spring and summer compared with autumn and winter and during the period SS compared with AW. Total volume was significantly higher in winter compared with all the other seasons; gel-free volume was higher in winter compared with summer and autumn. Mean sperm concentration was significantly lower in winter compared with spring and summer and in AW compared with SS. Total and progressive motility and membrane integrity did not show any significant difference between season and between SS and AW. A lower average path velocity was observed in autumn compared with spring and summer, and in summer a higher straight line velocity compared with spring and autumn, and a lower curvilinear velocity compared with winter were found. No differences in amplitude of lateral head displacement, beat cross frequency, straightness and linearity were observed neither between seasons nor in SS versus AW. Seminal pH did not show any seasonal difference; also sperm morphology did not show any significant difference during the whole year. This study demonstrated that under our climatic and management conditions Martina Franca donkey stallions do not show significant differences in testicular morphometric characteristics during the year, even if a certain seasonal influence exists on reaction time and some seminal parameters. Considering the variable temperature and photoperiod during the four seasons where the study was performed, this lack of substantial differences could be related to a characteristic of the Martina Franca breed.