Advances in Prostatic Diagnostics in Dogs: The Role of Canine Prostatic Specific Esterase in the Early Diagnosis of Prostatic Disorders.

Abstract In last years, following the increased canine life expectancy and the rising attention pet-owners devote to their animals, several authors have carried on investigations concerning new techniques to early identify canine prostatic disorders that might affect the dog's quality of life. Prostatic disorders often have an asymptomatic onset and their early diagnosis is difficult: hence, they are usually identified at an advanced stage, only. Traditionally, the diagnosis of prostatic disorders is based on noninvasive tools, such as transrectal and abdominal palpation, seminal or prostatic fluid evaluation, and urinalysis and imaging. On the other hand, a definite diagnosis of prostatic abnormalities could be achieved through prostatic parenchyma Fine Needle Aspiration (FNA) or biopsy. However, these investigations are performed rarely because of their invasiveness. Thus, several authors investigated canine serum biomarkers in order to achieve an earlier diagnostic timing and to apply therapeutic strategies for better outcomes. The Canine Prostatic Specific Esterase (CPSE) has been identified as a suitable biomarker to be included in a prostate health screening program, following the model of prostate-specific antigen (PSA) in human medicine. A higher CPSE in dogs suffering from several prostatic diseases, such as benign prostatic hyperplasia, bacterial prostatitis, or prostatic carcinoma, was reported in literature. Thanks to the potential usefulness in clinical practice, further studies should investigate the potential role of CPSE in monitoring the medical treatment success in the male reproductive system. Moreover, the spreading availability of serum biomarkers, easily carried out on blood samples in clinical practice, could assure a more accurate evaluation of the actual prevalence of prostatic disorders. The CPSE is actually recognized as a promising diagnostic tool for the detection of prostatic disorders in a "prostate health screening program," in order to properly select those patients requiring further more accurate and expensive diagnostic investigations

Differential surface glycoprofile of buffalo bull spermatozoa during mating and non-mating periods

The buffalo has a seasonal reproduction activity with mating and non-mating periods occurring from late autumn to winter and from late spring to beginning of autumn, respectively. Sperm glycocalyx plays an important role in reproduction as it is the first interface between sperm and environment. Semen quality is poorer during non-mating periods, so we aimed to evaluate if there were also seasonal differences in the surface glycosylation pattern of mating period spermatozoa (MPS) compared with non-mating period spermatozoa (NMPS). The complexity of carbohydrate structures makes their analysis challenging, and recently the high-throughput microarray approach is now providing a new tool into the evaluation of cell glycosylation status. We adopted a novel procedure in which spermatozoa was spotted on microarray slides, incubated with a panel of 12 biotinylated lectins and Cy3-conjugated streptavidin, and then signal intensity was detected using a microarray scanner. Both MPS and NMPS microarrays reacted with all the lectins and revealed that the expression of (i) O-glycans with NeuNAcα2-3Galβ1,3(±NeuNAcα2-6)GalNAc, Galβ1,3GalNAc and GalNAcα1,3(l-Fucα1,2)Galβ1,3/4GlcNAcβ1 was not season dependent; (ii) O-linked glycans terminating with GalNAc, asialo N-linked glycans terminating with Galβ1,4GlcNAc, GlcNAc, as well as α1,6 and α1,2-linked fucosylated oligosaccharides was predominant in MPS; (iii) high mannose- and biantennary complex types N-glycans terminating with α2,6 sialic acids and terminal galactose were lower in MPS. Overall, this innovative cell microarray method was able to identify specific glycosylation changes that occur on buffalo bull sperm surface during the mating and non-mating periods

Effects of intratesticular vs intraepididymal calcium chloride sterilant on testicular morphology and fertility in dogs.

Abstract Background Both stray and free-roaming owned dogs contribute to the serious global dog overpopulation problem. Many dog owners are unwilling to have their pet castrated for various reasons, including a reluctance to have their dog's behavior changed. A non-surgical method of sterilizing both stray and owned dogs would help to prevent unwanted litters. Previous studies have shown that intratesticular injection of calcium chloride dihydrate (CaCl2) in alcohol is a promising and cost-effective alternative to surgery for stray dogs, with testosterone significantly decreased and sexual activity eliminated. The aim of this study was to compare the use of a solution of 20% CaCl2 in 95% ethanol injected into the testicles or into the head of the epididymis. Methods A total of 148 dogs divided into 4 groups (2 experimental and 2 control) were respectively injected with CaCl2 or saline solution into the testicle or epididymal head (ultrasound-guided). The animals were examined at 0, 3, 6, and 9 months for sperm quality, concentration of testosterone in serum, and side effects; at 0 and 5 months with contrast-enhanced ultrasound (CEUS) to enhance the morphological aspects/alteration of the testicular parenchyma or epididymis; and at 9 months when all were castrated for histological examination. Results All dogs treated with CaCl2 became sterile with azoospermia achieved over the 9-month study. The concentration of testosterone in serum significantly decreased following intratesticular treatment with CaCl2. No adverse effects were noted. Conclusions A single, bilateral intratesticular injection of 20% CaCl2 in 95% ethanol was confirmed to be a reliable method for induction of sterilization in male dogs. The approach showed long-term efficacy and may reduce sexual behavior, with the additional benefits of low-cost and ease of use, making this nonsurgical method appropriate for use in stray dogs. Sterility was also achieved if injected in the head of the epididymis but no significant decrease in serum concentration of testosterone occurred. Moreover, performing the intraepididymal injection into the epididymal head was as time consuming as orchiectomy. This approach may be optimal for use in owned dogs where anatomical integrity and testosterone maintenance is preferred by the owner

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

<p>Abstract</p> <p>Background</p> <p>Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.</p> <p>Methods</p> <p>We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.</p> <p>Results</p> <p>We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.</p> <p>Conclusions</p> <p>The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.</p

Therapeutic Ultrasound as a Potential Male Dog Contraceptive: Determination of the Most Effective Application Protocol

Contents: Ultrasound is one of the most promising forms of non-invasive contraception and has been studied in several animal models. The objective of the current investigation was to determine the most practical and effective application protocol for dog sterilization. A total of 100 dogs were divided into five equal groups. Group A received 5-min applications three times performed at 48-hr intervals and covering the entire testicular area at frequency of 1 MHz; Group B received 5-min applications three times performed at 48-hr intervals over the dorso-cranial area of the testis at frequency of 3 MHz; Group C received three sequential 5-min applications (at 5-min intervals between applications) covering the entire testicular area at frequency of 1 MHz; Group D received 15-min applications two times performed at 48-hr intervals and covering the entire testicular area at frequency of 1 MHz. The experimental groups' ultrasound had an intensity of 1.5W/cm2. The Control Group had the same procedure as Group A, but with the transducer switched-off. Dogs were surgically castrated 40 days following the treatment for histological examination. Azoospermia, testicular volume reduction and apparently irreversible testicular damage were achieved by Group A. No effects were noticed in the other groups. Testosterone levels remained within physiological range with all application protocols. A regimen of three applications of ultrasound at 1 MHz, and 1.5 W/cm2, lasting 5 min with an interval of 48 h was effective as permanent sterilization in the dog without hormonal impact

Daily rhythms of behavioral and hormonal patterns in male dromedary camels housed in boxes

Background. Daily rhythmicity has been observed for a number of hormonal and behavioral variables in mammals. It can be entrained by several external factors, such as light-dark cycle and scheduled feeding. In dromedary camels, daily rhythmicity has been documented only for melatonin secretion and body temperature. In this study, the daily rhythmicity of behavioral repertoire, cortisol and testosterone levels was investigated in captive male camels. Methods. Six clinically healthy male dromedary camels (Camelus dromedarius) were used. The animals were housed in single boxes for 24 h daily and fed twice a day. Over a period of 48 h, behavioral observations were made and blood samples taken every two hours. The data were analyzed using diurnality index, conisor analysis and PROC mixed procedure. Results. The diurnality index for rumination and lying down was close to 0 (respectively, 0.09 and 0.19), while the indices for stereotypy, standing, feeding and walking were close to 1 (respectively, 0.74, 0.84, 0.92 and 0.85). Cosinor analysis revealed daily rhythmicity for all behaviors and for cortisol levels (acrophase at 12:57) but not for testosterone. Rumination and lying down (inactive behaviors) reached a peak during the scotophase, whereas feeding, walking and stereotypy (active behaviors) reached a peak during the photophase around midday. Cortisol level and expression of stereotypies peaked before and after food distribution and were negatively correlated (r =-0.287, P = 0.005). Testosterone levels and expression of sexual behaviors were stimulated by the visual and olfactory contacts with the females and were positively correlated (r =0.164, P =0.040). Testosterone was also negatively correlated with cortisol (r =-0.297; P =0.003). Discussion. These preliminary results provided new knowledge about the daily rhythm of behaviors in camels housed in boxes, suggesting that camels exhibit diurnal behavior pattern in the maintenance conditions outlined in the study. Daily rhythmicity seemed to be entrained not only by the light-dark cycle but also by scheduled feeding. The rise in stereotypy after food distribution could be due to the persistence of feeding motivation and frustration after the ingestion of food. Therefore, feeding practices should be improved to satisfy the foraging and feeding motivation of these camels. Behavioral and hormonal daily patterns in camels should be taken in consideration to adapt the management system, giving the animals more freedom during the light period and a diet richer in fiber, so as to improve reproductive performance, health and welfare

Mating behaviour and semen parameters in dromedary camel bulls (Camelus dromedarius): A comparison between two types of artificial vagina

Semen collection in the dromedary camel (Camelus dromedarius) is performed using different types of artificial vagina (AV) (long, short, with cervix imitation, with plastic or silicone inner liner) either held by an operator or fitted inside a camel dummy. However, systematic studies about the acceptance of such modifications by bulls, and their effects on sperm parameters, have yet to be investigated. This study was conducted to test the hypothesis that a silicone inner liner inserted into a standard cow AV could cause discomfort to camel bulls during mating, but at the same time could improve the quality of the ejaculates collected. Five bulls underwent semen collection sessions, with (n=3) and without (n=3) the silicone liner fitted in the AV, for a total of 30 semen collection sessions. A standard methodology and data-recording system were used for evaluating mating behavior, in addition to evaluation of semen parameters. There was no difference between an AV with or without silicone inner liner used during our standardized semen collection procedure. The use of such an AV modification hence still remains debatable, until further studies are able to identify any potential benefits of using this or other AV types or modifications. Such studies, however, should be performed by applying a standardized semen collection methodology and a proper evaluation of mating behaviour and semen parameters

Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential

BACKGROUND: Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle’s/Hank’s’ M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression. METHODS: Experiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls. RESULTS: EH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls. CONCLUSIONS: Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential