Effect of age and of reproductive status on reproductive indices in horse mares carrying mule pregnancies.

Mules (Equus mulus) are interspecific hybrids derived from crossing horse (Equus caballus) mares with jackasses (Equus asinus). In Italy, the production of mules is very important for the special environmental areas, where they are used as working animals, instead of the forbidden agricultural machines. Although many horse mares carry mule pregnancy successfully at term, low fertility of mares used for producing mules was reported, but limited data about reproductive efficiency in mares carrying mule pregnancy, especially when age and reproductive status are concerned, are available. Therefore, the present study aimed to assess the effect of age and of reproductive status on some reproductive indices of horse mares carrying mule pregnancy, in the final purpose to better clarify factors affecting reproductive performances in the valuable production of mules as working animals under special environmental areas in Italy. The study, performed on 96 draft mares inseminated with Martina Franca donkey semen, showed a satisfactory 89.6% 14 days PO pregnancy rate, lower pregnancy losses (19.8%) than those previously reported for mule pregnancies, leading to satisfactory foaling rates (71.9%), similar to those reported for intraspecific horse pregnancies. A clear, significant detrimental effect of mare's age on pregnancy losses, foaling rate, and on number of estrous cycles needed to achieve pregnancy, was found. Reproductive status also affected the reproductive indices, with significantly higher pregnancy losses and lower foaling rate in barren mares, reopening the question about the possible lower reproduction efficiency in horse mares carrying mule pregnancy. The present study results, although mostly descriptive, add useful information about some factors affecting reproductive performances in the valuable production of mules as working animals under special environmental areas in Italy.

Reproductive characteristics of foal heat in female donkeys.

In this study, the first postpartum heat, termed the foal heat, characteristics and performance in female donkey (jenny) of Martina Franca are described. To this end, the follicular development of 42 jennies during foal heat was compared with that of 31 jennies at the third estrus after foaling. Estrus length (7.1±0.9 and 6.8±0.7 days), follicular development and preovulatory follicle size (43.7±3.5 and 45.1±2.5 mm) were similar between jennies during the foal heat and during the third estrus after foaling. The pregnancy rate at day 14 was significantly lower in the foal heat jennies (57.1%) than the third estrus jennies (82.3%). However, the pregnancy rate at day 14 in foal heat jennies increased significantly when the onset of foal heat was ⩾8 days after foaling (93.8%) or when the ovulation happened ⩾12 days after foaling (85.7%). The data provided in the present study suggest that the foal heat in the endangered jennies of Martina Franca could be successfully utilized to reduce the interpartum period if the onset of foal heat occurs >8 days after foaling.

Correlation between some arterial and venous blood gas parameters in healthy newborn Martina Franca donkey foals from birth to 96 hours of age.

In neonatology, blood gas analysis is a useful tool in the evaluation of the health of newborns and plays a key role in early detection of critically ill subjects. Because blood gas analysis parameters have not previously been studied in any depth in donkey foals, this study was performed on 16 healthy Martina Franca donkey foals born after an uncomplicated delivery. Arterial and venous blood samples were collected at 5 minutes and at 12, 24, 72, and 96 hours of age. Blood gas analysis was performed by a portable analyzer, measuring arterial and venous total carbon dioxide, carbon dioxide partial pressure (pCO2), oxygen partial pressure (pO2), oxygen saturation (sO2), bicarbonate, base excess (BE), pH, and lactate (LT). Lower blood pH values, pO2 and sO2, and a higher level of lactate were found at birth in comparison with subsequent sampling times. This moderate acidotic profile disappeared at 12 hours, when all the parameters became constant until the end of the study period. As expected, significant differences between arterial and venous blood gas parameters related to the oxygenation, such as pO2 and sO2, and partially carbon dioxide partial pressure were found, whereas total carbon dioxide, pH, BE, and LT were comparable in arterial and venous blood samples. For these latter parameters, the highly significant correlation between arterial and venous findings suggests that venous samples could be an acceptable alternative to the arterial sample for blood gas analysis in newborn donkey foals, when the oxygenation status of the patient is not the first goal of patient analysis.

Transrectal ultrasonographic evaluation of combined utero-placental thickness during the last half of pregnancy in Martina Franca donkeys.

In the recent years, the donkey population decreased dramatically so that many breeds are presently considered as endangered. In comparison to the horse, the donkey placenta still remains not completely studied. In the horse, one of the diagnostic tools useful to identify pregnant mares at risk of abortion or premature delivery, include the transrectal ultrasound examination of the uterus and its contents; and especially of the combined thickness of the uterus and of the placenta (CUPT). Since the CUPT was never investigated in donkeys, the present study was aimed to define the transrectal CUPT values during the last half of pregnancy in 20 Martina Franca jennies. Foalings times, foals characteristics and placental gross appearance, and measurements were also evaluated and values resulted always within normality. Differently to the mare, a continuous significant CUPT increase between the sixth to the 12 months of pregnancy, and a substantial increase from the ninth to the 12th month of pregnancy, was found. Although statistically not evaluable, the CUPT values recorded from three jennies with pregnancy loss did not show evidence of CUPT increases. In conclusion, normal CUPT values from the sixth to the 12th month of pregnancy in Martina Franca donkeys are provided, but further investigations are needed to define possible breed or body-size CUPT specific differences, as well as the CUPT values during pregnancy disturbances or placental abnormalities.

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-17ß (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.

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.

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.

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.

Influence of mycotoxins on spontaneous contraction in myometrial strips of prepubertal lamb.

The effects of mycotoxin zearalenone and their major metabolites alpha- and beta-zearalenol on spontaneous contractions in isolated lamb uterine smooth muscle were examined. The study was carried out on 20 female prepubertal lambs aged between 45 and 50 days. Myometrial strips were set up in two isolated organ baths (10ml) at 37 degrees C and were exposed to increasing concentrations (10(-11)M-10(-6)M) of these mycoestrogens and results were compared with the effect, at the same concentrations, of natural estrogen 17beta-estradiol. Our findings suggest that mycotoxins and 17beta-estradiol, at nanomolar concentrations, rapidly enhance phasic spontaneous smooth muscle contraction. In particular, zearalenone increases the uterine activity similarly to 17beta-estradiol. On the contrary, its metabolite alpha-zearalenol significantly inhibits myometrial contractility.

Plasma homocysteine levels in cycling, pregnant, and spayed bitches.

The aim of this study was to evaluate the physiological range of homocysteine (Hcy) exhibited by bitches during the follicular (pro-oestrus), luteal (dioestrus) and anoestrus phases of cycling animals, and in pregnant and spayed bitches. The lowest concentrations of Hcy were observed during dioestrus (3.2+/-1.27micromol/L) and in pregnant bitches (3.9+/-1.72micromol/L), and the highest concentrations during anoestrus (7.8+/-0.6micromol/L) and in spayed bitches (12.1+/-5.16micromol/L).

Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices.

We studied the roles of mGlu2/3 receptors (mGlu2/3) in glutamatergic transmission at corticostriatal synapses in mice brain slices. Perfusion of the selective mGlu2/3 agonists LY354740 and L-CCG1 caused the long term depression (LTD) of evoked synaptic responses. Photonic and electronic microscopy showed mGlu2/3 on axonal fibers and glial processes but not on striatal dendrites. mGlu2/3-LTD was independent of synaptic activity and insensitive to specific antagonists of dopamine D1, D2, GABA(B), N-methyl-D-aspartate or adenosine A1 receptors. Manipulation of the cAMP/protein kinase A cascade had no effect on the mGlu2/3-LTD. In contrast, MEK1-2 inhibitors reduced both mGlu2/3 initial depression and LTD suggesting the involvement of the mitogen activated kinase pathway in mGlu2/3-LTD.

Localization and mechanisms of action of cannabinoid receptors at the glutamatergic synapses of the mouse nucleus accumbens.

Despite the role of excitatory transmission to the nucleus accumbens (NAc) in the actions of most drugs of abuse, the presence and functions of cannabinoid receptors (CB1) on the glutamatergic cortical afferents to the NAc have never been explored. Here, immunohistochemistry has been used to show the localization of CB1 receptors on axonal terminals making contacts with the NAc GABAergic neurons. Electrophysiological techniques in the NAc slice preparation revealed that cannabimimetics [WIN 55,212,2 (WIN-2) and CP55940] strongly inhibit stimulus-evoked glutamate-mediated transmission. The inhibitory actions of WIN-2 were dose-dependent (EC(50) of 293 +/- 13 nm) and reversed by the selective CB1 antagonist SR 141716A. In agreement with a presynaptic localization of CB1 receptors, WIN-2 increased paired-pulse facilitation, decreased miniature EPSC (mEPSC) frequency, and had no effect on the mEPSCs amplitude. Perfusion with the adenylate cyclase activator forskolin enhanced glutamatergic transmission but did not alter presynaptic CB1 actions, suggesting that cannabinoids inhibit glutamate release independently from the cAMP-PKA cascade. CB1 did not reduce evoked transmitter release by inhibiting presynaptic voltage-dependent Ca(2+) currents through N-, L-, or P/Q-type Ca(2+) channels, because CB1 inhibition persisted in the presence of omega-Conotoxin-GVIA, nimodipine, or omega-Agatoxin-IVA. The K(+) channel blockers 4-aminopyridine (100 micrometer) and BaCl(2) (300 micrometer) each reduced by 40-50% the inhibitory actions of WIN-2, and their effects were additive. These data suggest that CB1 receptors are located on the cortical afferents to the nucleus and can reduce glutamate synaptic transmission within the NAc by modulating K(+) channels activity.

A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells.

We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.