In vitro and in vivo effect of oxytetracycline on sperm parameters in breeding rooster.

BACKGROUND: Some antimicrobials could adversely affect sperm quality during sperm cryopreservation and antibiotic treatment with subsequent effects on fertility outputs. To our knowledge, no similar studies have been conducted on breeding roosters, especially for oxytetracycline (OTC). OBJECTIVE: To investigate both in vitro and in vivo impact of oxytetracycline on sperm parameters in breeding roosters. METHODS: Sperm motility parameters were objectively analyzed using the CASA system including total motility (TM %), progressive motility (PM %), all sperm velocities, the sperm count, and cell viability during 9 days of in vivo treatment. In the in vitro investigation, the pooled sperm was diluted and divided into a control aliquot (diluted in 0.9% NaCl) and treated samples. Motility parameters were assessed after 0, 1, 2, 3, 4, 5, and 6 hours of storage at 37ºC. In the in vivo study, 1 g per L of OTC was administrated to five individuals for nine consecutive days. Fresh semen samples were analyzed at T0 (before treatment) and after 6 (T6) and 9 days (T9) of treatment. RESULTS: OTC caused significant impairment of sperm quality in vivo. A drastic reduction in sperm concentration, viability, TM, PM, and all kinematic parameters was observed after 6 days of treatment. However, at day 9 sperm quality had improved to be nearly similar to T0. In vitro, OTC induced similar sperm impairment on all sperm motility parameters. CONCLUSION: Oxytetracycline exhibited negative effects on rooster sperm both in vivo and in vitro and appears consequently not suitable in cryopreservation extenders. Doi.org/10.54680/fr23510110412.

Influence of the coincubation of post-thawed bull semen with elevated ß-hydroxybutyrate concentrations on sperm characteristics.

In this study, the relationships between post-thaw bull sperm characteristics and hyperketonemic conditions after coincubation with cow plasma or media were determined to investigate if such a condition could affect bull sperm characteristics. Two experiments were conducted. In experiment 1, blood samples were collected from 31 cows to prepare plasma. Cows were independently categorized into two groups according to plasma ß-hydroxybutyrate (BHB) concentrations (above or below 1.2 mM). Thawed bull semen was diluted and incubated with diluted plasma; motility parameters were evaluated using Computer Assisted Semen Analysis (CASA). In experiment 2, a pooled sample of thawed semen was diluted and divided into three aliquots: without BHB (control) and treated with either 1.2 mM (1.2) or 3 mM (3) BHB. In addition to motility, flow cytometric analyses were carried out. In experiment 1, the overall motility decreased significantly in plasma containing high (≥1.2 mM) BHB compared to plasma containing low (<1.2 mM) BHB. In experiment 2, the overall motility tended to be lower in BHB (3 mM)-supplemented samples. The supplementation of 3 mM BHB increased the proportion of live superoxide-positive sperm and sperm with high mitochondrial potential, while the DNA fragmentation index decreased.

Camptothecin: solubility, in-vitro drug release, and effect on human red blood cells and sperm cold preservation.

BACKGROUND: Camptothecin (CPT) is an anticancer drug, and is not employed in the clinic because of its high hydrophobicity and low active form stability. CPT may also have potential for use in cold preservation. OBJECTIVE: To overcome these drawbacks, CPT solubility variations in the presence of cyclodextrins (CDs) and polyethylene glycol (PEG) were evaluated by Higuchi solubility experiments. MATERIALS AND METHODS: CPT was encapsulated in different cyclodextrins and polyethylene glycol using a co-evaporation method. The CPT interactions with CDs and PEG 6000 were investigated by Fourier-transformed infrared spectroscopy (FT-IR), and X-ray powder diffraction (XRPD). Then, CPT complexes were evaluated for in-vitro drug release. To evaluate the potential anticancer efficacy of the CPT complexes system, in-vitro cytotoxicity studies on human red blood cells were carried out using UV assay. The impact of the CPT complex systems on sperm motility protection during cold preservation at 4 degree C was studied using CASA. RESULTS: The dissolution profile of these preparations shows the improvement of the dissolution of the CPT following a fickien diffusion. The CPT solubility and stability improvement were the cause of the cytotoxicity on the red blood cells test. However, CPT alone, encapsulated, dispersed, and chemically modified protected spermatozoids during cold preservation. CONCLUSION: We confirm the interest in CPT encapsulated and dispersed in anticancer treatments. We also found that CPT encapsulated or dispersed could protect sperm against oxidative damage and improve the membrane integrity of human sperm. Consequently, CPT encapsulated our dispersed could eventually be beneficial for infertility therapy. Doi: 10.54680/fr23210110712.

Drug release, stability and efficiency of vitamin e loaded in liposomes for bovine sperm protection in cryopreservation medium.

BACKGROUND: It is known that a considerable number of drugs in clinical use or under development are water-insoluble drugs with poor bioavailability. The liposomal delivery system has drawn attention as one of the noteworthy approaches to increase both dissolution and absorption because of its biocompatibility and ability to encapsulate hydrophobic molecules in the lipid domain. However, several drawbacks have been reported, the most common is liposome structural instability. OBJECTIVE: To encapsulate alpha tocopherol into liposomes, to determine the new formulation stability and to study the drug-release of alpha tocopherol into the sperm cryopreservation medium. MATERIALS AND METHODS: The liposomes prepared by an ethanol injection method were characterized for size stability, alpha tocopherol release and sperm motility tests. RESULTS: The prepared unilamellar vesicles had both narrow size distribution (around 99 nm) and a good physical and chemical stability at 4°C during 12 months. The liposomes did not release the vitamin E immediately, but retained the protectant for 24 hours, probably due to the rigidity of the liposomal fence which was reinforced by adding cholesterol. Then, all vitamin E molecules were released by 48 hours. Release was potentially by Fickian diffusion probably by the creation of mini-ducts due to both agitation and fence hydration. Moreover, semen motility treated with vitamin E liposome preparations was significantly improved compared to all other treatments (including commonly used sperm conservation media). CONCLUSION: The stable vitamin E liposomes formulated in this work are a promising alternative for semen cryopreservation protection.

Encapsulation Nanotechnology in Sperm Cryopreservation: Systems Preparation Methods and Antioxidants Enhanced Delivery.

Sperm cryopreservation promotes the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. However, sperm cryopreservation causes several stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Supplementing cryoprotectant media with antioxidants has been reported to be positive in different species. It has been widely suggested to combine antioxidants with nanotechnology, to maximize therapeutic activity and to minimize undesirable side effects. In this review, we discuss the role of different antioxidants in sperm cryopreservation and their improved therapeutic effect through their formulation using nanotechnology. In addition, we report the different nano-systems preparation methods present in literature. Whilst the use of nanotechnology in animal production is still in its infancy, encouraging results from nutrition, biocidal, remedial, and reproductive studies are driving further investigations.

Rosmarinus officinalis Essential Oil Preloaded in ß-Cyclodextrin: Effect on Ram Spermatozoa Motility, Membrane Integrity and Oxidative Status During 4°C Storage.

BACKGROUND: Rosmarinus officinalis essential oil (Rom) has been reported recently to be of interest for use in sperm cryopreservation. However, related to its lipophilic characteristics, encapsulation in cyclodextrin could enhance Rom positive effects by increasing its solubility in sperm extenders. OBJECTIVE: To compare the effect of Rom preloaded in hydroxypropyl-ß-cyclodextrin (Rom-cd) to Rom alone (Rom) on ram sperm conserved at 4°C. MATERIALS AND METHODS: Ram epididymal sperm was collected from six testes. Each collected sperm was split into four equal aliquots. The control aliquot was diluted with Tris extender (Tris + citric acid + fructose + penicillin), two aliquots were treated with Rom at 0.5µl ml-1 and 1µl ml-1 respectively, and one aliquot treated with Rom-cd at 1µl ml-1. All sperm aliquots were analyzed for motility after 0, 2, 4, 24 and 48 h of storage at 4°C using a Computer Aided Semen Analysis (CASA). Membrane integrity and oxidative stress status were measured after 48 h of storage. RESULTS: The results indicated that motility parameters were best preserved in the extender containing Rom-cd compared to the groups treated by Rom without cyclodextrin. Rom alone resulted to higher sperm motility than the control group. Lower oxidative stress and more cell membrane protection were observed in Rom treated samples, especially when using Rom-cd. CONCLUSION: The ability of Rom to protect ram sperm against cryopreservation damages was improved after encapsulation in hydroxypropyl-ß-cyclodextrin (HPßCD).

Effect of the essential oil of Rosmarinus officinalis (L.) on rooster sperm motility during 4°C short-term storage.

AIM: This study aimed to investigate the protective effect of Rosmarinus officinalis (L.) essential oil on rooster sperm motility during 4°C short-term storage. MATERIALS AND METHODS: R. officinalis essential oil was analyzed using gas chromatography coupled to mass spectrometry to identify the active components. 10 of 45-week-old Hubbard commercial broilers were subjected to biweekly semen collections during 3 weeks. At each collection, sperm was pooled and divided into four aliquots and then diluted with Tris extender supplemented with 870, 87, or 8.7 µg/ml of R. officinalis essential oil, identified as treatments R, R5, and R10, respectively. Tris-based extender without any supplementation was considered as a control group. Diluted sperm was then stored at 4°C in the refrigerator and analyzed at 0, 6, 24, and 48 h using a computer-assisted sperm analyzer. Different semen parameters were measured including total motility, progressive motility, gametes velocities (straight line velocity [VSL], curvilinear velocity [VCL], and average path velocity [VAP]), amplitude of the lateral head displacement [ALH], and beat-cross frequency [BCF]. RESULTS: The phytochemical analysis of R. officinalis essential oil revealed the presence of 25 active components including seven major molecules: Camphor (18.88%), camphene (5.17%), 1,8-cineole (7.85%), ß-thujene (13.66%), α-thujene (4.87%), chrysanthenone (12.05%), and ß-cubenene (7.97%). The results showed a beneficial effect of R. officinalis essential oil on sperm cells motility, particularly when using the lowest concentrations, 8.7 and 87 µg/ml. Progressive motility and gametes velocities (VCL, VSL, and VAP), materializing the quality of gametes motility, showed highly statistically significant values (p<0.01) in 8.7 and 87 µg/ml treatments, especially from 6 h of storage at 4°C. Conversely, the highest concentration (870 µg/ml) showed harmful effects with a total spermicidal activity after 24 h of storage. CONCLUSION: The current results revealed the positive impact of R. officinalis essential oil on rooster sperm at 4°C short-term storage probably through fighting against oxidative stress and cold shock damages.

The Complementary Effect of Cholesterol and Vitamin E Preloaded in Cyclodextrins on Frozen Bovine Semen: Motility Parameters, Membrane Integrity and Lipid Peroxidation.

BACKGROUND: During cryopreservation sperm cells suffer from two major deleterious impacts: oxidative stress and cold shock. OBJECTIVE: To investigate in bovine species the benefit of cholesterol and vitamin E, both loaded in cyclodextrins, as a double protection against oxidative stress and cold shock. MATERIALS AND METHODS: Semen was collected from nine mature bulls using an artificial vagina and each ejaculate was split into four equal aliquots. The control aliquot was diluted with Fraction A (Tris+citric acid+fructose+penicillin) without further supplementation; the treated samples were diluted in Fraction A supplemented with cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) or CD-CHL in association with CD-Vit E (CD-CHL-VitE). After incubation at 22°C for 15 min and addition of Fraction B (Fraction A+egg yolk+glycerol), all aliquots were frozen in 0.25 ml straws. Straws were then thawed individually at 37C for 30 seconds in a water bath and immediately analyzed for motility, using Computer Aided Semen Analysis (CASA), membrane integrity and oxidative stress status. RESULTS: The results showed that samples treated with CD-CHL and CD-Vit E were protected against the deleterious impact of freezing thawing process. However, the optimal protection was observed when the two complexes CD-CHL and CD-Vit E were simultaneously used. All analysed semen parameters including motility, membrane integrity and oxidative stress status were significantly improved in CD-CHL-Vit E compared to all other treatments. CONCLUSION: Cholesterol and vitamin E, both preloaded in cyclodextrins to increase their solubility, appeared as a powerful protection in cryopreserved bovine semen to fight simultaneously cold shock and oxidative stress.

Effects of female bovine plasma collected at different days of the estrous cycle on epididymal spermatozoa motility.

The aim of this study was to assess the effects of female bovine plasma collected at different days of the reproductive cycle on epididymal spermatozoa motility and to test hypothesis that the subpopulations pattern of motile spermatozoa is affected by this treatment. Blood plasma samples were collected from five Holstein Friesian cows at different stages of the estrous cycle (days 0, 5, 10, 12 and 18), one pregnant cow and one adult bull and were diluted 1:9 (V/V) with normal saline. Female charcoal-treated plasma, Bull plasma and saline were used as controls. Semen samples were obtained from cauda epididymidis through retrograde flushing and diluted in saline to approximately 60 × 106 sperm/ml. The extended semen was diluted 1:2 (V/V) with tested media and motility was evaluated at 15 min and then every hour for 6 h using a computer-assisted semen analysis. Multivariate clustering procedure was applied to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into three separate subpopulations with defined patterns of movement: Subpopulation 1 poorly motile and non-progressive spermatozoa (39.3%), subpopulation 2 including the fastest and the most vigorous spermatozoa (46.4%) and subpopulation 3 represented by slow, non-vigorous but linear spermatozoa (14.3%). Initially, sperm samples supplemented with female, male or female charcoal-treated plasma stimulated equally total motility and spermatozoa belonging to subpopulation 2 regardless of the estrous cycle stage. After 1-h incubation, the motility of these both categories of spermatozoa (total motile and those assigned to subpopulation 2) is enhanced and maintained more in day 12, 18 and pregnant cow plasma than in female plasma from earlier stage of the estrous cycle (day 0, 5 and 10), male plasma and female-charcoal treated plasma. In conclusion, the overall results showed that female plasma stimulated significantly sperm motility, especially at the late stage of the estrous cycle. Additionally, to the diverse compounds contained in blood plasma, progesterone may play a key role in such motility activation.

Presence of antimicrobial resistance in coliform bacteria from hatching broiler eggs with emphasis on ESBL/AmpC-producing bacteria.

Antimicrobial resistance is recognized as one of the most important global health challenges. Broilers are an important reservoir of antimicrobial resistant bacteria in general and, more particularly, extended-spectrum ß-lactamases (ESBL)/AmpC-producing Enterobacteriaceae. Since contamination of 1-day-old chicks is a potential risk factor for the introduction of antimicrobial resistant Enterobacteriaceae in the broiler production chain, the presence of antimicrobial resistant coliform bacteria in broiler hatching eggs was explored in the present study. Samples from 186 hatching eggs, collected from 11 broiler breeder farms, were inoculated on MacConkey agar with or without ceftiofur and investigated for the presence of antimicrobial resistant lactose-positive Enterobacteriaceae, particularly, ESBL/AmpC-producers. Escherichia coli and Enterobacter cloacae were obtained from the eggshells in 10 out of 11 (10/11) sampled farms. The majority of the isolates were recovered from crushed eggshells after external decontamination suggesting that these bacteria are concealed from the disinfectants in the egg shell pores. Antimicrobial resistance testing revealed that approximately 30% of the isolates showed resistance to ampicillin, tetracycline, trimethoprim and sulphonamides, while the majority of isolates were susceptible to amoxicillin-clavulanic acid, nitrofurantoin, aminoglycosides, florfenicol, neomycin and apramycin. Resistance to extended-spectrum cephalosporins was detected in eight Enterobacteriaceae isolates from five different broiler breeder farms. The ESBL phenotype was confirmed by the double disk synergy test and blaSHV-12, blaTEM-52 and blaACT-39 resistance genes were detected by PCR. This report is the first to present broiler hatching eggs as carriers and a potential source of ESBL/AmpC-producing Enterobacteriaceae for broiler chicks.

Correlation of five radioimmunoassay systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancy period.

The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them ( > or = 0.81; P<0.001). PAG concentrations increased significantly from Day 21 (n=27) to 30 (n=37) after AI by use of all PAG-RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B(0) binding to the tracer. Our results suggest that all tested PAG-RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.

Comparison of five radioimmunoassay systems for PAG measurement: ability to detect early pregnancy in cows.

This study was conducted to describe the minimum detection limit, reproducibility, accuracy, specificity and parallelism of different pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) systems: RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool. Their ability to distinguish between non-pregnant and pregnant females at day 30 after artificial insemination (AI) was investigated. The antisera were raised in rabbits against different PAG preparations. All RIA systems proved to be sensitive, repeatable and accurate for measuring PAG concentrations. The dilutions of plasma samples taken at an early stage of pregnancy were found to be parallel to the standard curves. No cross-reaction was observed with different carbohydrates, either with Pregnant Mare Serum Gonadotropin (PMSG) or human Chorionic Gonadotropin (hCG). The concentrations of PAG in pregnant females at day 30 after AI were shown to be higher with the use of antisera R#706, R#780, R#809 and Pool when compared with antiserum R#497. All the RIA systems gave 100% sensitivity and negative predictive values. On the other hand, the use of antisera R#780 and R#809 resulted in lower specificity and positive predictive values. The present study clearly shows that the ability of PAG-RIA systems to diagnose pregnancy specifically at day 30 after AI can be improved by using a combination of antisera raised against different forms of PAG.

Influence of progesterone concentrations on secretory functions of trophoblast and pituitary during the first trimester of pregnancy in dairy cattle.

The essential role played by progesterone in the maintenance of pregnancy is unequivocal; however, the effects of progesterone on the secretory patterns of placental and pituitary molecules during the gestation period are not well defined. The objective of this study was to describe pregnancy-associated glycoprotein (PAG) concentrations (measured by RIA-497 and RIA-Pool) in pregnant females with progesterone concentrations lower (low-P4 group, n=20) or higher (high-P4 group, n=17) than the mean of 8.74 ng/mL on Day 21 (AI=Day 0). Luteinizing hormone (LH) and prolactin concentrations were also measured in both groups. Throughout the study period, blood samples were collected on Days 0, 21, 45, 60, and 80 from 37 females that were confirmed to be pregnant. PAG concentrations measured by both RIA-497 and RIA-Pool tended to be higher in high-P4 group than in low-P4 group from Day 30 until Day 80. On Day 80, plasma PAG concentrations that were measured using RIA-497 were observed to be higher (P<0.05) in the high-P4 group than in the low-P4 group (10.2+/-8.7 ng/mL versus 6.9+/-3.8 ng/mL). Concentrations of LH on Day 60 and prolactin on Day 80 were observed to be significantly lower (P<0.05) in the high-P4 group. There was a tendency for the concentrations of LH (Days 45 and 80) and prolactin (Days 30, 45, and 60) to be lower in cows in the high-P4 group than in the low-P4 group. Our results suggest the existence of a relationship among the concentration levels of progesterone, PAG, LH, and prolactin during early pregnancy.

Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: in vitro and in vivo studies.

The effects of medium exchange on motility parameters of chilled canine semen preserved in egg yolk Tris-glucose (EYTG) extender were analyzed over a 27-d period. Semen extender was exchanged at three time points (Days 11, 21 and 27) after collection, when motility parameters were demonstrated to significantly decrease from parameters observed at semen preparation (Day 0) or at day of previous extender exchange. In the absence of medium exchanges, motile spermatozoa were observed up to Day 16 (mean +/- S.D. 1.5 +/- 0.3% of motile spermatozoa). A stimulation of the different semen motility parameters was observed after extender exchange. Semen extender exchange at Day 11 allowed conservation of motility until Day 21, compared to 16 d in the absence of extender exchange. At Day 21, when spermatozoa appeared immobile or dead, a second extender exchange was performed, allowing the extension of motility conservation up to Day 27. The third extender exchange, performed at Day 27, was no longer associated with motility stimulation. Glucose content in the medium decreased slowly over time; a concomitant decrease in pH was also observed. No changes in osmolarity were observed over time. To verify the fertility of long-term conserved chilled semen, two groups of 10 bitches were inseminated either once (Group 1) or twice at 48-h intervals (Group 2) intra-vaginally with semen conserved chilled for a mean of 9 +/- 1.8 d. Out of the 10 bitches inseminated once, 5 became pregnant, versus 7 in the group of animals inseminated twice. The present study reports the possibility to extend the conservation of chilled canine semen up to 3 wk with conservation of good fertility for at least 10 d. The role of energetic substrate and pH alteration is postulated and the classically accepted relation of semen motility/viability is raised.

Computer assisted semen analyzers in andrology research and veterinary practice.

The evaluation of sperm cell motility and morphology is an essential parameter in the examination of sperm quality and in the establishment of correlations between sperm quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective assessment of different cell characteristics: motion, velocity, and morphology. The development and problems related to this technology are raised in this review, paying particular attention to the biases and standardization requirements absolutely needed to obtain useful results. Although some interesting results, mainly in humans, have already been obtained, many questions remain, which have to be answered to allow for further development of this technology in veterinary medicine, clinical fertility settings, physiological, and toxicology research activities. The main problem is related to the standardization and optimization of the equipment and procedures. The different CASA instruments have all demonstrated high levels of precision and reliability using different sperm classification methodology. Their availability gives us a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa.

Validation of the sperm quality analyzer (SQA) for dog sperm analysis.

In the present study, a simple and inexpensive unit (the Sperm Quality Analyzer-SQA), was evaluated for dog sperm analysis. Our objective was to propose a cheap, accurate and convenient device to be used in veterinary practices involved with dog fertility assessment and artificial insemination. The device was tested by analyzing repeatability and accuracy at different sperm concentrations and motility characteristics. The Sperm Motility Index (SMI), a numeric index provided by the SQA, was compared with the results obtained using a computer-aided sperm analyzer (Hamilton Thorn IVOS 10). The correlation between SMI and some sperm parameters as well as predictive values of the SMI were established. The dog sperm data provided by the SQA were consistent and repeatable (coefficient of variability below 10% for all concentrations tested). The SMI was significantly dependant on motile sperm concentration and a positive significant correlation was established for the different motile sperm concentrations from a concentration of 25 x 10(6) up to over 200 x 10(6) cells/mL. Zero motility did not affect SMI because non-motile cells, regardless of their concentration, do not cause any fluctuations in the optical density (OD). Over the tested 200 x 10(6) cells/mL value, a correlation still could be observed but it was not statistically significant, possibly because of a saturation of the system. In dog semen, the correlation is better between SMI values and the number of motile spermatozoa than with the overall motile concentration. Based on this observation, a predictive value was given to the SMI allowing for a sorting of dog ejaculates in 3 sperm categories (SMI <100, 100250) each characterized by a range of sperm number and motility. If a positive correlation between the SMI categories and fertility has been demonstrated in humans, such a correlation needs to be established in dogs.

Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders.

The present study was conducted to evaluate chilled semen conservation over time in 3 commercial and 4 laboratory prepared extenders, including a new Tris-glucose extender. The beneficial effect of adding egg yolk to these media was also analyzed. The effects of these extenders on motility and acrosome reaction were characterized objectively using a computer-aided semen analyzer and the chlortetracycline staining, respectively. No significant differences were observed when comparing the different commercial extenders without egg yolk, but addition of egg yolk improved all motility parameters significantly (preservation of 50% of motility was observed at 3.2+/-1, 2.9+/-0.5, 2.3+/-0.5, 8.5+/-0.2, 5.4+/-1.1, 5.2+/-0.4 d, for Biladyl, green extender and fresh-phos extenders without and with egg yolk, respectively). Motility parameters were best preserved in egg yolk supplemented Biladyl extender with a mean percentage of 86.3+/-10.5 motile spermatozoa after 7 d at 4 degrees C. Efficacy of egg yolk-supplemented commercial extenders on sperm motility at 4 degrees C was (in decreasing order) as follows: Biladyl > green extender > fresh-phos. However, high quality motility and the percentage of motile spermatozoa were highest with some of the laboratory prepared extenders: a 50% conservation rate of motile spermatozoa was observed following the use of supplemented egg yolk extenders. These are classified in decreasing order as follows: Tris-glucose (13+/-1 d) > Tris-fructose (9.7+/-0.6) > EDTA (4.+/-0.6 d) > Tris-bes (3.6+/-1.1 d). A low concentration of motile spermatozoa was still observed in the Tris-glucose egg yolk extender 16 d after collection, clearly demonstrating the importance of the medium and the beneficial effect of egg yolk on sperm motility of 4 degrees C chilled semen. Similar effects of extender were observed for acrosome reactions. Egg yolk clearly had a protective effect reducing acrosome reactions significantly in all media tested as follows: the highest acrosome losses were observed in the fresh-phos and EDTA extenders without egg yolk; the lowest rate was observed with Tris-glucose supplemented egg yolk extender. In conclusion, at 4 degrees C, egg yolk extender best-protected sperm motility parameters. Differences in osmolarity between the extenders in terms of substrate related to sperm metabolic activity may explain the optimal results obtained using egg yolk-supplemented Tris-glucose extender, which preserved motility and acrosome integrity in chilled dog semen. These results indicated that good quality dog spermatozoa could be preserved for up to 10 d.

Evaluation of the "Hamilton Thorn computer-based automated system" for dog semen analysis.

An objective evaluation of semen is warranted to assess the canine male fertility and to select appropriate techniques and extenders for semen preservation. With conventional microscopic evaluation, the subjectivity of the analysis makes any comparison of results difficult. In the present study, we validated the Hamilton Thorn computer-aided semen analyzer (HTR-IVOS10 analyzer) for objective assessment of canine semen. A description of fertile canine motility parameters using this analyzer is reported. Semen analysis at 38 degreesC is found to be more optimal and accurate than 30 degreesC. The Makler chamber was preferred to the Cell-vu, which induced a decrease of all semen motility parameters. The repeatability of the measures was good with intra-and inter-assay coefficients of variation below 10% and 20%, respectively, for most of measured parameters. An overestimation of semen concentration, increasing with dilution of semen, was observed when HTR-IVOS10 results were compared with the classical manual Makler cell evaluation. A significant decrease in semen motility parameters was recorded when high semen dilutions were used. Generated from the analysis of 42 mature fertile male beagle dogs, a description of semen motility parameters using the CASA system is presented to serve as reference when comparing Beagle ejaculates both in clinical and experimental studies.

Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste.

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.

Effect of finasteride (Proscar MSD) on seminal composition, prostate function and fertility in male dogs.

This study reports the long-term effects and reversibility of the administration of Finasteride, a 5 alpha-reductase inhibitor, on the prostate, prostatic fluid composition and volume, sperm output and characteristic, and fertility of adult beagle dogs treated for 21 weeks at a dose of 1 mg kg-1 body weight. Finasteride was not associated with any side effects. After 5 to 15 weeks of treatment, it induced a marked decrease in the size of the prostate and a fall in its secretions and in the production of spermatozoa. Sperm concentration increased and was inversely correlated with the decrease in the volume of prostatic secretions. At maximum effect, the calculated prostate volume was reduced to 30% of the initial value, and together with the reduced prostatic secretion, no ejaculate could be collected despite normal behaviour. These effects were reversible in 6 to 8 weeks after the cessation of treatment. Matings at 20 to 22 weeks after the start of treatment were fertile, demonstrating the absence of long-term effects of this treatment on male fertility. As fertility was not impaired and prostatic benign hyperplasia successfully regressed. Finasteride treatment in this preliminary study appears to be an interesting alternative therapy in valuable breeding dogs with benign prostatic hyperplasia.