Heat SHOCK proteins in equine spermatozoa: Expression and correlation to kinetic and environmental parameters.

Heat Shock Proteins are chaperones primary involved in the repair of cellular damages induced by temperature. The harmful effect of temperature on the male gonad is well known, on the contrary knowledge on the effects of the environment on semen quality are still insufficient. The aim of this paper was to learn more about the role of HSPs and the environment in modulating the physiology of equine male gonads. We showed a detailed analysis of equine semen characteristic and the expression level of three HSPs (60-70-90) over a one-year period analyzing the effects of temperature and humidity and the correlation among the different variables. We showed also that the interpretation of results depends strongly on the way in which data are assembled and analyzed, therefore we compared results obtained from three different ways of grouping: according to single months, to weather seasons and to mare reproductive periods. Results showed that the expression of the three HSPs is correlated to the environment through temperature and humidity and that it reaches the highest level in the breeding season and in summer. We found also that HSPs expression is correlated to some variables describing the quality of equine semen (concentration) and the kinetic of spermatozoa (total motility-MOT, %, average path velocity -VAP, µm/s- and lateral head displacement -ALH, µm). No correlation was found between HSPs expression and the mitochondrial membrane potential; while viability and HSP90 expression resulted positively correlated. The month-by-month analysis evidenced that in February equine semen has the highest kinetic characteristics (increased linearity -LIN, %-, straightness -STR, % -and average path velocity -VAP, µm/s) with the highest number of motile, progressive motile and rapid cells. These results may have a great impact in the comprehension of functional aspects of the physiology of equine semen and may have potential implications for breeders who want to understand the period (and/or month) of the year in which equine semen reaches the best characteristics with increased chances for better results in reproductive practice.

Assessment of a natural dietary extract, titrated in phenylpropanoid glycosides, on blood parameters and plasma oxidative status in intensively reared Italian hares (Lepus corsicanus).

Two different doses of a natural extract titrated in phenylpropanoid glycosides (PPGs) were evaluated for their effect on blood parameters and plasma oxidative status in pairs of intensively reared Italian hares. The study lasted 210 days, during which 45 couples of hares were divided into three homogeneous groups. A control group (CON) was fed a control diet while the two experimental groups were fed a diet supplemented with 1 or 2 kg/t of a supplement titrated in PPGs. Blood samples were obtained at 0, 70, 140 and 210 days and assayed for plasma lipid profiles, bilirubin, haematological parameters and indicators of oxidative status (reactive oxygen metabolites (ROMs), thiobarbituric acid reactive substances (TBARS), vitamins A and E). Although dietary treatment did affect the levels of triglycerides, total cholesterol and total bilirubin, all of which decreased markedly (P < 0.05), while significantly increasing the (P < 0.01) HDL cholesterol values, it also significantly improved the oxidative status of the blood, which displayed an increase in both vitamin E (P < 0.01) and vitamin A (P < 0.05) and a decrease in ROMs (P < 0.01) and TBARS (P < 0.05). The improvements in the blood parameters, lipid profile and plasma oxidative status continued to increase significantly as the trial progressed, indicating a positive effect with increased length of treatment. The results of this study demonstrate an important role for feed supplementation with respect to antioxidant activity on some blood parameters, including the lipid profile and the oxidative status of blood.

Induction of fertile oestrus in cats by administration of hCG and calcium-naloxone.

The aim of this study was to evaluate the possibility of inducing fertile oestrus in queens by administering hCG in combination with Ca(2+)-naloxone. It is well established that an increase in endogenous opioids leads to a decrease in LH. The administration of naloxone, an opioid antagonist, inhibits endogenous opioidergic tone and induces the onset of pro-oestrus. The opioidergic block is related to the increase in binding of beta-endorphins to specific receptors, which determines calcium channel blockage. Pretreatment with hCG results in a rapid increase in the number of LH receptors and Ca(2+)-naloxone induces G protein activity. Twenty-one anoestrous queens were divided into four groups: (i) group 1, nine queens were treated with a single s.c. injection of hCG (1000 iu) and daily for 4 days with 0.1 ml kg-1 body weight i.m. of a solution containing 0.4 mg naloxone ml-1 dissolved in 20% calcium gluconate; (ii) group 2, four animals were treated with a single s.c. injection of hCG (1000 iu); (iii) group 3, four queens were treated with Ca(2+)-naloxone (0.1 ml body weight kg-1 i.m.) daily for 4 days; and (iv) group 4, four queens received no treatment (controls). Queens were monitored using vaginal cytology and blood progesterone concentrations, and pregnancy was detected using ultrasonography. In groups 2, 3 and 4 clinical signs of oestrus were not observed. In group 1, 88.8% of treated queens were mated (8 of 9) and ovulated on the basis of an increase in progesterone, and 75% (6 of 8) of these queens became pregnant. In conclusion, pretreatment with hCG increased the number of LH receptors and Ca(2+)-naloxone antagonized the hypothalamic GnRH opioid block thus inducing the pulsatility of LH leading to fertile oestrus in queens.

In vitro maturation and fertilization of follicular oocytes in cattle.

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.