In vitro antioxidant activity of the prostatic secretory granules in rabbit semen after exposure to organic peroxides.

BACKGROUND: The prostate gland of rabbits produces numerous granules, which are specifically implicated in the inhibition of sperm capacitation during the first hours after mating. These granules are rich in vitamin E, but their role in the antioxidant protection of rabbit sperm has not been studied. AIM OF STUDY: The objectives of this study were to investigate whether the prostatic secretory granules (PSGs) could prevent sperm induced-lipid peroxidation and to verify a potential involvement of tocopherols from the PSGs to the sperm. METHODS: Washed sperm samples taken from eighteen White New Zealand rabbits were either incubated with tert-butyl-hydroperoxide (t-BHP, an oxidative stressor) or with buffered Tyrode's medium for 1 hour. The same number of sperm samples that contained PSGs were subjected to the previously mentioned treatments and thiobarbituric acid reactive substances (TBARS), vitamin E compounds and the acrosome status were assessed. RESULTS: The incubation of the sperm with t-BHP resulted in a noticeable production of TBARS (0.38 vs. 0.22 nmol/10(7) cells) and an associated decrease of alpha-tocopherol (alpha-T, 72.3 vs. 103.2 nmol/10(8) cells) with respect to the sperm samples containing PSGs. The sperm incubated with the PSGs had a higher amount of alpha-T compared to the control (292.2 vs. 251.0 nmol/10(8) cells). The acrosome status was not affected by the occurrence of the organic peroxide in the medium and the amount of capacitated sperm was lower when the PSGs were also present. CONCLUSIONS: Overall, these results suggest that the PSGs may represent a source of protection for rabbit sperm against in vitro oxidative stress by supplying the sperm with endogenous alpha-T. This mechanism could be in part involved in the inhibition of sperm capacitation by the granules.

In vitro toxic effects of metal compounds on kinetic traits and ultrastructure of rabbit spermatozoa.

Metal compounds have been associated with male reproductive toxicity in vivo. The aim of the present study was to investigate the in vitro effects of 20 metal compounds using rabbit ejaculated spermatozoa as a study model for spermiotoxicity. Five of the metals tested (arsenic, cadmium, chromium, mercury and vanadium) reduced sperm motility and curvilinear velocity. Ultrastructural analyses revealed three types of damage to sperm head membranes in relation to the metal used: acrosome breakage with formation of various sized microvesicles (arsenic, cadmium, mercury and platinum); a large round hole (arsenic, cadmium and chromium), and numerous folds in the acrosome membrane (vanadium). The vanadium compound, followed by chromium and mercury compounds, determined a higher number of damaged spermatozoa. In conclusion, all the studied metal compounds, at levels higher than 1microM, may reduce sperm kinetic characteristics and probably fertilizing capacity by triggering specific morphological damages to the head and/or by inhibiting motility.

Lipid composition of the main fractions of rabbit semen.

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 microg/10(9) but was only 21-23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp(p)) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE+PC ratios than in the germinal cells. Some physiological implications are discussed.

Effect of chocolate and Propolfenol on rabbit spermatogenesis and sperm quality following bacterial lipopolysaccharide treatment.

The aims of the study were to evaluate the effects of chocolate and propolis-enriched diets on rabbit spermatogenesis, sperm motility, and ultrastructure following bacterial lipopolysaccharide (LPS) treatment. Thirty-two New Zealand White rabbits were divided into four groups. The LPS-Propolfenol(®) group received propolis (500 mg/kg/day) in their diet for 15 days, while the LPS-chocolate group was fed 70% cacao chocolate (1 g/1 kg/day) for the same period. Following the diet treatments, rabbits in the LPS-Propolfenol(®) and LPS-chocolate groups, and an LPS group received a single intraperitoneal dose of 50 µg/kg LPS, and the control group received only saline. Kinematic sperm traits were evaluated with a computer assisted sperm analyzer (CASA) system, and ultrastructural characteristics were examined by transmission electron microscopy (TEM). Testicular and epididymal tissues were observed by light microscopy and TEM and multiplex real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect and quantify toll-like receptor-4 (TLR-4) gene expression. The values of the analyzed semen parameters of rabbits treated with LPS-Propolfenol(®) and LPS-chocolate did not show any variations compared with the control group, but they were lower in rabbits treated only with LPS. Alterations observed in the testicular tissue of LPS treated-rabbits were not detected in specimens from the LPS-chocolate and LPS-Propolfenol(®) groups, which showed normal spermatogenesis. The TLR-4 mRNA expression was similar in controls, in LPS treated, and in LPS-chocolate groups, but it was significantly (p < 0.01) decreased in LPS-Propolfenol(®) rabbits. In conclusion, a chocolate and propolis-enriched diet showed a protective effect on the spermatogenetic process of buck rabbits following LPS treatment.

Role of rabbit prostate granules on sperm viability and acrosome reaction evaluated with different methods.

In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO(2). Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating.

Desmosterol, the main sterol in rabbit semen: distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility.

Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form (94.3%), were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol (58.5% of total sterols) and cholesterol (35.9% of total sterols). Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% (in the prostatic secretory granules, PSGs) to 63.8% (in the seminal plasma). Spermatozoa contained an intermediate proportion of desmosterol (59.8%), which was asymmetrically distributed between the heads (52.0% of the total content of sterols) and the tails (81.8%). Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L(-1). In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation.

The main factors affecting the reproductive performance of rabbit does: a review.

This paper reviews the main factors affecting the reproductive performance of rabbit does. In the last 15 years the profitability of rabbit farms has increased mainly due to improvements in management and genetic selection but several problems related to animal welfare have also occurred. The replacement and the mortality rates of female per year are very high and the replaced does often show poor body condition and poor health status. The effect of kindling order, litter size, genetic strain, weaning age and reproductive rhythm on the reproductive performance and welfare of females and mechanisms implicated in these effects are discussed. The rabbit doe of modern strains produces a lot of milk with high energetic value, which leads to a mobilization of body fat resulting in a negative energy balance. In the current reproductive rhythms, there is an extensive overlap between lactation and gestation. The resulting energetic and hormonal antagonism reduces the fertility rate and lifespan of the doe. Strategies to optimize these parameters are discussed. An approach that combines various strategies seems to be required to meet these objectives. Since the factors involved in this system are predetermined (genetic strain, environment) the most powerful way to improve doe welfare is to choose a reproductive rhythm more adapted to the physiology of the does.

Long term effect of post-weaning rhythm on the body fat and performance of rabbit doe.

Reproductive protocols based on standard (Control: 11 days post-partum) or extended rhythm (PW: post-weaning at 27 days post-partum) were compared. Two groups of fifty 19-week-old New Zealand White females were inseminated for seven consecutive cycles. The kits were weaned at 26 days. On the day of AI, all the does were submitted to ultrasound scanning of the perirenal regions to measure fat thickness. The fertility rate and several indexes of efficiency were calculated. Fat thickness, estimated perirenal fat and live weight were higher in PW does. The does submitted to post-weaning rhythm had a higher sexual receptivity (P < 0.01), and fertility rate (P < 0.01) whereas litter size and pre-weaning mortality were not affected. Primiparous Control does showed a particularly low fertility rate; the value increased successively but was always lower than in PW does. PW rhythm in comparison with the standard one seemed more adapted to doe reproductive physiology even if there was a lower production (35.0 vs. 38.8 rabbit sold/year) and risk of fatness (18% of multiparous does).