BACKGROUND: Canine overpopulation is a global issue with serious health and welfare implications. Nonsurgical methods of sterilization could yield positive impacts on this problem, but no long-term data on such methods are available. The objective of the current investigation was to determine the effects of intratesticular injections of calcium chloride dihydrate (CaCl2) in saline in dogs over a one year period. Five concentrations (0%, 10%, 20%, 30%, 60%) of CaCl2 in saline were administered via intratesticular injection to groups of 10 dogs each. Total sperm count and motility, blood levels of testosterone, and side effects were examined at 0, 2, 6, and 12 months post-injection (PI). Testicular size and semen volume were examined at 0 and 12 months PI. RESULTS: Total sperm count, semen volume and testosterone showed significant dose-dependent decreases upon treatment with 10%-60% CaCl2 compared with either the control group (0% CaCl2) or baseline for each treatment group. Azoospermia was achieved for at least 12 months PI in 60% and 80% of treated dogs after administration of a 10% and 20% CaCl2, respectively. Treatment with 30% or 60% CaCl2 resulted in azoospermia in 100% of dogs, but more side effects were observed, while no side effects were noticed at lower doses. For each treatment group, testosterone levels had decreased an average of 35%-70% at 6 months following treatment. However, testosterone levels rebounded by the 12-month time point in all groups except the highest dosage group (60% CaCl2), which remained at the low end of physiological range throughout the study. Sperm motility dropped to zero or near zero in all dogs treated with CaCl2. Testicular size was significantly smaller at 12 months PI for all groups when compared to baseline. CONCLUSIONS: This first long-term study confirms reports of the efficacy of CaCl2 sterilization. However, at dosages free of adverse events, calcium chloride in saline may not provide permanent sterilization as previously believed. Future work should explore optimized solvents to increase the permanence of the well-tolerated 20% formulation.

Background: Surgical castration is widely used to sterilize male dogs, but has significant impacts on time to perform the operation, recovery of the animals as well as cost, which can limit population control programs. Previous research has shown intratesticular injection of calcium chloride dihydrate (CaCl2) in saline to be a promising alternative to surgery. However, long-term azoospermia was not maintained at dosages low enough to avoid side effects. In the search for an optimized formulation, the current investigation is the first study on long-term sterilization effects of intratesticular injection of CaCl2 in either lidocaine solution or alcohol in dogs. CaCl2 at 20% concentration in lidocaine solution or alcohol was administered via intratesticular injection to groups of 21 dogs each. The treated animals were examined at 2, 6, and 12 months for sperm production, blood levels of testosterone, and side effects; at time zero and 12 months for testicular size and semen volume. The experimentally treated animals were compared to a control group receiving saline injection only. Results: Testicles of dogs treated with CaCl2 in either diluent significantly decreased in size. After administration of CaCl2 in lidocaine solution, sterility was achieved for at least 12 months in 75% of treated dogs. However, optimal long-term contraceptive effectiveness was achieved with CaCl2 in alcohol, which resulted in azoospermia over the 12-month study period. Testosterone levels significantly decreased following treatment with CaCl2, and sexual activity disappeared. Although testosterone returned to baseline levels by 12 months for the group treated with CaCl2 in lidocaine, dogs injected with CaCl2 in alcohol had a 63.6% drop in testosterone level, which remained at the low end of physiological range throughout the study. No adverse effects were noted. Conclusions: A single, bilateral intratesticular injection of 20% CaCl2 in 95% ethanol was a reliable method for induction of sterilization in 18–28 kg male dogs in this study. The approach showed long-term efficacy and reduced sexual behavior. This chemical method of sterilization might provide an effective, efficient alternative to surgical castration that can have positive impacts on dog welfare.

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or ;1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

Methodologies, such as flow cytometry and computer assisted sperm analysis (CASA), provide objective, reproducible, rapid and multi-parametric evaluation of semen quality. In this study, semen samples collected from six stallions were analysed for viability (by propidium iodide), chromatin stability by sperm chromatin structure assay (SCSA) and mitochondrial membrane potential by JC-1 using flow cytometry. Total and progressive motility, average path velocity (VAP), curvilinear velocity (VCL) and straight-line velocity (VSL) were determined by CASA system. The cytofluorimetric analysis provided results with low intra-assay variability respect to motility analysis by CASA system. The data on viability and mitochondrial assessment were rather uniform between stallions. The SCSA was able to distinguish potential fertility levels between stallions. In fact statistical differences were found between stallions especially for %-DFI and SD-DFI parameter. The %-DFI parameter was negatively correlated with VCL parameter. The higher repeatability of %-DFI parameter respect to those of other SCSA parameters confirms the importance of this parameter notoriously related to fertility. In conclusion, the simultaneous assessment of different functional sperm parameters, by flow cytometry and CASA, may be allow to obtain detailed and repeatable evaluations of sperm quality in the stallion, usually not considered in breeding selection programs.

Due to the increased attention that pet-owners devote to their animals and to the improved veterinary care, investigations regarding methods to early detect prostatic disorders that might affect canine life quality have been performed. Canine prostate specific esterase (CPSE) concentration was reported to be higher in dogs suffering from prostatic diseases. This study aimed to estimate the CPSE threshold as a biomarker to early identify prostatic diseases in asymptomatic dogs. The ultrasonographic examination of the prostate was performed in 19 dogs (6–40 kg; 1–5 years) with no symptoms of prostatic diseases. Dogs were grouped according to the presence (Group A) or absence (Group B) of prostatic disorders at the ultrasound (altered appearance, the presence of cysts or irregular borders). For each dog, a venous blood sample was collected to measure serum CPSE and the ratio between calculated and normal expected prostatic volume was assessed for each dog. The CPSE data were statistically analysed (t test, p < .05), and the CPSE threshold in blood serum between groups was calculated by ROC. In 11 dogs, ultrasonography showed signs of prostatic abnormalities (Group A, 2–5 years), while no signs were detected in eight dogs (Group B, 1–3 years). The calculated/estimated volume ratio resulted greater than 1.5 in Group A dogs. The CPSE was statistically different between groups (p < .0001): higher in Group A (mean = 184.9, SD = 126 ng/ml) than in Group B (38.9 ± 22.1 ng/ml). The cut-off CPSE threshold was 52.3 ng/ml (ROC, AUC = 0.974, SE 95.6%, SP 89.2%). This study suggests that CPSE serum concentration higher than 50 ng/ml in asymptomatic dogs is associated with ultrasonographic alterations and increased the prostatic size (volume by 1.5 times greater than the normal size). As the onset of prostatic disorders often remains asymptomatic, the rapid assessment of CPSE could be suitable for selecting preventively those animals that would require further accurate evaluation.

An affordable and effective non-surgical technique for achieving male dog sterility is needed to solve the problem of overpopulation. The efficacy of 20% calcium chloride in pure alcohol solution, injected into the testicular parenchyma, as a method for chemical castration, was evaluated. Twenty-one dogs of mixed breed, 4.7 ± 1.23 years old, 20 ± 5.84 kg of body weight, with good clinical conditions and normal reproductive parameters, were lightly sedated and injected into the dorsocranial portion of both testes with a solution of 20% calcium chloride dihyrdate in ethanol (95%). The dose injected corresponds with the testicular width (19–22 mm receive 0.8 ml; 23 and above 1 ml). Semen evaluation was performed by CASA (Computer Assisted Sperm Analysis) system at day 30–60–90. The animals in the control group received a single bilateral intratesticular injection of 1 ml sterile saline solution (testicular width 23 mm and above). Forty-eight hours after the injection, dogs showed very light discomfort at palpation and testicular tumefaction, which regressed within 3 days. At day 30, testicular ultrasonography revealed bilateral more dense nodular lesions; prostatic volume and parenchyma were normal. Semen evaluation showed azoospermia at day 30–60 and 90. The sperm count was decreased significantly (p < 01) in all the CaCl2 treated dogs in comparison to saline solution control animals. At day 90 testicles were shrunk at palpation. An intratesticular injection of 20% calcium chloride in pure alcohol solution, as a method for chemical castration, was effective and economical for the sterilization of male dogs. It is free from pain and chronic stress and will contribute to a simple alternative method to surgical castration. The dogs of this study are under evaluation to study this solution long term effect (1 year).

Background: Reproductive biotechnologies in dromedary camel (Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate the bioenergy/oxidative status of dromedary camel oocytes before and after IVM by confocal microscopy 3D imaging. Methods: Oocytes were retrieved by slicing ovaries collected at local slaughterhouses. Recovered oocytes were examined before and after IVM culture for nuclear chromatin configuration and bioenergy/oxidative status, expressed as mitochondria (mt) distribution and activity, intracellular Reactive Oxygen Species (ROS) levels and distribution and mt/ROS colocalization. Results: The mean recovery rate was 6 oocytes/ovary. After IVM, 61% of oocytes resumed meiosis and 36% reached the Metaphase II stage (MII). Oocyte bioenergy/redox confocal characterization revealed changes upon meiosis progression. Immature oocytes at the germinal vesicle (GV) stage were characterised by prevailing homogeneous mt distribution in small aggregates while MI and MII oocytes showed significantly higher rates of pericortical mt distribution organized in tubular networks (P<0.05). Increased mt activity in MI (P<0.001) and MII (P<0.01) oocytes compared to GV stage oocytes was also observed. At any meiotic stage, homogeneous distribution of intracellular ROS was observed. Intracellular ROS levels also increased in MI (P<0.01) and MII (P<0.05) oocytes compared to GV stage oocytes. The mt/ROS colocalization signal increased in MI oocytes (P<0.05). Conclusions: This study provides indications that qualitative and quantitative indicators of bioenergy and oxidative status in dromedary camel oocytes are modified in relation with oocyte meiotic stage. These data may increase the knowledge of camel oocyte physiology, in order to enhance the efficiency of IVP procedures.

In: Reproduction, Fertility and Development, Vol 25(1), 2013 Juvenile in vitro embryo transfer in farm animals reduces the generation interval and increases the rate of genetic gain. In human reproductive medicine, it enables the preservation of female fertility of young patients affected by cancer or by premature ovarian failure. The developmental competence of in vitro-produced juvenile embryos is strictly related to oocyte quality. The aim of the present study was to analyse the developmental potential and the mitochondrial/oxidative status of ovine prepubertal oocytes matured in vitro to clarify their suitability in juvenile in vitro embryo transfer programs. Oocytes from the ovaries of slaughtered prepubertal lambs (less than 6 months of age) were analysed after in vitro maturation. After cumulus cell removal, oocytes at the metaphase II stage (MII) underwent either IVF plus in vitro embryo culture (Experiment 1; n 1⁄4 200; Bogliolo et al. 2011 Reprod. Fert. Dev. 23, 809–817) or confocal analysis of mitochondria (mt) and reactive oxygen species (ROS) fluorescence distribution, intensity, and colocalization (Experiment 2; n 1⁄4 30; Martino et al. 2012 Fertil. Steril. 97, 720–728) or scavenger enzyme [superoxide dismutase (Ambruosi et al. 2011 PLoS ONE 6, e27452) and catalase (Beers and Sizer 1952 J. Biol. Chem. 195, 133–140)] activity analyses in cell lysates of individual oocytes (Experiment 3; n 1⁄4 7). In Experiment 1, 150 of 200 MII oocytes (75%) cleaved after 30 h of in vitro embryo culture, and 36 of 150 2- to 4-cell-stage embryos (24%) reached the blastocyst stage at Day 8. In Experiment 2, 60 of 111 (54%) oocytes selected for in vitro maturation culture reached the MII stage, and 30 of them (50%) with a regular size (.150 mm in diameter) and morphology were analysed for bioenergy/redox parameters. Fourteen of 30 oocytes (47%) showed a heterogeneous (perinuclear, pericortical, or both) mt distribution pattern, and the remaining 16 of 30 oocytes (53%) showed a homogeneous distribution of small mt aggregates. Intracellular ROS were uniformly distributed, thus not corresponding to the mt distribution pattern. Fluorescent intensity of mt and ROS labelling, expressed as arbitrary densitometric units, were 821.4 ` 274.7 and 737.6 ` 226.5 in oocytes with a heterogeneous pattern and 723.7 ` 371.6 and 831.7 ` 263.7 in oocytes with a homogeneous pattern, respectively (not significant). The mt/ROS colocalization (Pearson correlation coefficient) did not differ between heterogeneous (0.47`0.2) and homogeneous (0.51`0.09; not significant) oocytes. In Experiment 3, superoxide dismutase (n 1⁄4 4) and catalase activity (n 1⁄4 3) values were 1.09 ` 0.03 and 10.63 ` 1.96 IU mg1 of protein, respectively. This study provides basal values of bioenergy/redox parameters in prepubertal lamb MII oocytes as related to their developmental potential and may increase the knowledge of prepubertal oocyte physiology compared with their young adult counterparts.

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.

Camel management has been changing in recent years from an extensive to a semi-intensive or intensive system, particularly for breeding bulls and dairy dromedary camels. Captivity may affect animal welfare, and low libido is the major complaint for housed breeding bulls. Since welfare status could also affect reproductive performance, the aim of this study was to evaluate different management practices on behavior, particularly on sexual behavior, and to identify some behavioral needs of male dromedary camels reared for semen collection. The effects of the following management systems on their behavior were compared: i) traditional: housing in a single stall for 24 hours (H24), ii) housing in a single stall for 23 hours with one hour free in the paddock (H23) and iii) housing in a single stall for 22 hours and 30 min with 1 h paddock time and 30 min exposure to a female camel herd (ExF). During the trial, blood cortisol concentrations were assessed and camels were filmed daily for thirty minutes in the mornings and during a female passage in the evenings. Videos were analyzed in order to fill out a focal sampling ethogram and to score sexual behavior. As a result, there were no differences between the H24 and H23 systems, whereas ExF had a significant positive impact on their sexual behavior score and behavioral repertoire, further reducing cortisol levels. Overall, it seems that male dromedary camel welfare status improves when their behavioral needs for social interaction and movement are satisfied.

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.

The oviduct plays an essential role in the mammalian reproduction and it undergoes significant endocrine-induced morphological, biochemical and physiological changes during the oestrous cycle. The functions of the oviduct epithelium are controlled by the ovarian steroids, oestrogen and progesterone1. In this study the glycoconjugate pattern of oviduct obtained from hormonally (FSH-P and eCG) superovulated ewes for oocytes recoverywas analyzed. Oviducts from treated and control sheep were collected by laparatomy, fixed in 4% (w/v) neutral formalin, embedded in paraffin wax and sections processed for lectin histochemistry. In the ampulla, the luminal surface of all specimens showed strong reactivity with MAL II,SNA, PNA after KOH-sialidase (s) treatment, RCA120, HPA, SBA, KOH-s-WGA, and GSA I-B4, whereas it stained strongly with GSA II, UEA I, and LTA in treated sheep which showed reactivity with KOH-s-PNA, SBA, GSA I-B4, GSA II also in the apical cytoplasm of non-ciliated cells. In the isthmus, the luminal surface showed same staining reactivity with RCA120, SBA, and GSA I-B4 in all specimens, and a stronger affinity for MAL II, UEA I and LTA in treated ones. A distinctive feature of hormonized isthmus was the binding of the entire cytoplasm of ciliated cells and non-ciliated cells with MAL II, SNA, RCA120, SBA, GSA II, UEA I, and LTA. These results indicate that ampulla and isthmus of ovine oviduct express a different glycoconjugate pattern and that the hormone administration for superovulation produces different effect along the oviduct. These differences could be related to the different functions of each segment that constitutes the ovine oviduct. 1. Buhi WC Reproduction 2002, 123:355-62.

BACKGROUND. Benign prostatic hyperplasia (BPH) is a result of urogenital aging. Recent studies suggest that an age-related impairment of the blood supply to the lower urinary tract plays a role in the development of BPH and thus may be a contributing factor in the pathogenesis of BPH. The canine prostate is a model for understanding abnormal growth of the human prostate gland. We studied the efficacy of pulsed electromagnetic field therapy (PEMF) in dogs to modify prostate blood flow and evaluated its effect on BPH. METHODS. PEMF (5 min, twice a day for 3 weeks) was performed on 20 dogs affected by BPH. Prostatic volume, Doppler assessment by ultrasonography, libido, semen quality, testosterone levels, and seminal plasma volume, composition and pH were evaluated before and after treatment. RESULTS. The 3 weeks of PEMF produced a significant reduction in prostatic volume (average 57%) without any interference with semen quality, testosterone levels or libido. Doppler parameters showed a reduction of peripheral resistances and a progressive reduction throughout the trial of the systolic peak velocity, end-diastolic velocity, mean velocity, mean, and peak gradient of the blood flow in the dorsal branch of the prostatic artery. The pulsatility index and the resistance index did not vary significantly over time. CONCLUSIONS. The efficacy of PEMF on BPH in dogs, with no side effects, suggests the suitability of this treatment in humans and supports the hypothesis that impairment of blood supply to the lower urinary tract may be a causative factor in the development of BPH. Prostate 74:1132–1141, 2014. # 2014 The Authors. The Prostate published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The optimization of the semen collection procedure and the extension of the breeding season are important issues for improve camel reproductive management. GnRH treatment has been suggested to increase male libido in many species, but achieved results are still conflicting in dromedary camels. The aim of this work was to investigate the effect of a single GnRH analogue dose (100 μg of gonadorelin, Fertagyl®) on testosterone level, behavioral and semen parameters. The same camel bulls (n=5) were used as control (C) and experimental (E) group, and the GnRH was administered trice every 48 hours. Blood samples were collected by catheter every 20 minutes, from 2 p.m. to 6 p.m. (T0 - T12) and semen collection procedures started soon after sampling. GnRH did not affect the testosterone basal level (T0, C vs E: 3.0±0.8 vs 3.5±0.9 ng/ml; P=0.94) and no effect of the repetition, was assessed (P=0.41). Testosterone level showed an upward trend only in treated animals, became statistically significant after 1 hour (T3, C vs E: 2.7±0.7 vs 8.8±2.6 ng/ml; P=0.02), peaking after 140 minutes (T7, C vs E: 2.8±1.8 vs 11.9±3.8 ng/ml; P=0.007), and then slowly decreasing (T12: C vs E: 3.6±1.1 vs 8.7±1.6 ng/ml; P=0.02). During the semen collection procedures the bulls showed a tendency increase in libido, reporting a significant rise in sperm concentration (C vs E: 1085.18±139.6 vs 491.26±139.6 106/ml; P=0.02). From our data, the tested GnRH therapy may be suggested to enhance dromedary male’s libido and probably the optimal time window to collected semen should be between 2 and 3 hours after its administration. So, GnRH effects on behavioral and seminal parameters need to be ascertained with further investigations during more appropriate timing.

The purpose of this studywas to assess the natural exposure ofmale horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of theDNA fragmentation index (DFI), such as the mean (¯X DFI), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, a-zearalenol, and b-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized. # 2010 Elsevier Inc. All rights reserved.

Postpartum uterine diseases, mainly metritis, are very common in dairy cows which exhibit sickness, reduced milk yield and reproductive disorders. Puerperal metritis causes severe economical loss due to costs for treatment, milk withdrawal, reduced fertility and premature culling. Early detection of animals at risk for metritis remains a challenge for veterinarians. The aim of this study was to understand the relationship between variation of haematochemical and metabolic parameters and postpartum metritis in order to evaluate if some parameters could be used as predicting factors. Fifty Italian Fresian cows of an intensive dairy farm were submitted to blood sampling on d -45 ~ (Dry period), d -15 ~ (Close-up period) and d +15 ~ (Postpartum period) for determination of haematochemical profile, serum protein profile, Non-Esterified Fatty Acids (NEFA) and β-Hydroxybutyrate (BHB). Immediately after delivery 4 cows died thus were not considered in the study. All cows were daily examined and divided into 2 Groups according to puerperal discharge: presence of normal and odorless lochia (healthy=19); presence of fetid red-brown or white purulent discharge with or without signs of systemic illness (sick=27). All data were statistically analysed (P<0.01) using the General Linear Model (SAS®, Cary, NC, USA). No significant difference (P>0.05) between the 2 Groups in Dry period was detected. Considering the Close-up period, significant differences between the 2 Groups (healthy vs sick) as regards Hb (g/dl) (10.24±0.19vs11.17±0.13, P<0.01); HCTm (%) (28.40±0.57vs30.86±0.40, P<0.01); HCT (%) (28.60±0.54vs31.45±0.38, P<0.01); α1 globulins (%) (8.34±0.39vs6.06±0.28, P<0.01); WBC (103/μl) (6.99±0.51vs8.77±0.36, P<0.01) and NEFA (mEq/lt) (0.13±0.06vs0.35±0.04; P<0.01), were found. In the postpartum period significant differences were found as regards α2 globulins (%) (7.62±0.26vs8.52±0.18; P<0.01) and NEFA (mEq/lt) (0.39±0.08vs0.66±0.06; P<0.01). These observations underline the importance of nutrition in modulating the innate immune system, in transition period, which in turn could play a role on the emergence of uterine diseases. Our results indicate that monitoring some defined parameters in transitional cows could be useful to early detection of animals at risk for postpartum uterine diseases.

Camels are seasonal breeders, and their sexual behavior is influenced by environmental conditions, but the relationship between climatic factors and sexual behavior has been poorly described in the available literature. Nowadays, the male camel living habit is shifting towards captivity; thus, this study was carried out to evaluate the sexual behavior of housed male dromedary camel through female’s parades and to correlate it with climatic parameters. Four housed sires, reared for semen collection, and one dam were used and the trial lasted 8 weeks, considering the first week as control. Six days per week and during evenings, the female was brought near each males’ boxes, while two observers filled a behavioral sampling ethogram and scored the male sexual behavior. After this parade, blood samples were taken from the female to evaluate the estradiol concentration. In addition, the following meteorological parameters were recorded, everyday, at 9:00 a.m. and 19:00 p.m.: pressure, wind, temperature, humidity, and H-index. The correlation between sexual behavioral score and female estradiol concentration and climate parameters was analyzed. All the behavioral parameters showed a significant upward trend; female estradiol concentration varied during the period and picked at week 5. Male sexual behavior was negatively correlated with morning H-index, wind, and temperature, and positively correlated with pressure and evening humidity, whereas it was not correlated with estrogen. In conclusion, female parade was a successful method to evaluate and stimulate the occurrence of housed male dromedary camel sexual activity that resulted to be negatively affected by hot temperature, warm wind, and lack of rain.

Ochratoxin A (OTA) is a renal mycotoxin and transplacental genotoxic carcinogen. The aim of this study was to evaluate the natural occurrence of OTA in equine blood samples and its placental transfer. For the assessment of OTA levels, serum samples were collected from 12 stallions, 7 cycling mares and 17 pregnant mares. OTA was found in 83% of serum samples (median value = 121.4 pg/mL). For the assessment of placental transfer, serum samples were collected from the 17 mares after delivery and from the umbilical cords of their foals, after foaling. Fourteen serum samples from pregnant mares contained OTA (median value = 106.5 pg/mL), but only 50% of their foals were exposed (median values = 96.6 pg/mL). HPLC analysis carried out on four serum samples (collected from two mares and their respective foals) supported the ELISA results on OTA placental transfer. This is the first report on the natural occurrence of OTA in horse serum samples and placental transfer in horses.

Background: The present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. Methodology/Principal Findings: A large (.8 mm in diameter) and a small (,8 mm) cell line were cultured in medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 mM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 mM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca2+]o and/or NPS R-467 reduced doubling time values. Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. Conclusions/Significance: In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

The oviduct isthmus is considered to be a sperm reservoir prior to ovulation in the reproductive tract of mammals. Ovarian steroids regulate the synthesis and secretion of specific molecules such as glycoproteins that are involved in the interactions between germ cells or embryos and oviductal epithelial cells. The objective of the present study was to investigate the effects on histmus glycoprotein pattern from hormonally stimulated ewes by means of lectin histochemistry. Isthmus fragments were separated from oviducts immediately after laparatomy, and, after fixed in 4% (w/v) neutral formalin, they were embedded in paraffin wax. Then, the sections were stained with a panel of lectins which revealed: 1) an increase of reactivity with MAL II, SNA, RCA120, SBA, GSA I-B4, GSA II, UEA I and LTA in the whole cytoplasm of ciliated and non-ciliated cells of hormonally treated females, 2) a reduction of DBA affinity in the luminal surface, 3) no staining pattern modification with PNA, KOH-sialidase (s)- PNA, HPA, Con A and KOH-s-WGA. These results indicate that exogenous gonadotropin administration for superovulation may alter the oligosaccharidic composition of glycoproteins produced in the ovine isthmus.

Abstract The aim of the present study was to assess the effects feeding level on body weight changes and semen parameters in adult Sardinian rams reared under intensive conditions in a semi-arid area of southern Italy. During an experimental period of 90 days, 24 healthy Sardinian rams were divided into three equal groups that differed in their feeding level, in terms of concentrate amount. The controlconcentrate (CC; n=8) group received 1.0 times their maintenance requirements, the medium-concentrate (MC; n=8) group received a diet that supplied 1.2 times their maintenance requirements, and high-concentrate (HC, n=8) group received a diet that supplied 1.5 times their maintenance requirements. Mixed vetch–oat hay was offered ad libitum to ram groups and water and mineral licks were freely available. Body weight and feed intake was recorded weekly, and semen characteristics were determined every 2 weeks. Dietary treatment affected final body weight (P<0.01) as feeding level increased. Total dry matter and protein intake changed significantly (P<0.05 and P<0.01, respectively) among experimental groups. Semen volume and concentration were positively influenced by feeding level for HC group, whereas no differences were observed in sperm viability and scrotal circumference of rams. It was concluded that dietary level with higher concentrate supplementation resulted in improved body weight gain, feed intake, sperm production, and semen quality in Sardinian rams.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

Stallion sperm from semen collected in Southern Italy during the breeding (June–July) and non-breeding (December–January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc, with Galb1,3GalNAc, a/bGalNAc and glycans with terminal/internal aMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galb1,4GlcNAc (Ricinus communis120 affinity) (RCA120) and LFuca1,2Galb1,4GlcNAcb (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and postacrosomal region did not display glycans terminating with GalNAc, GlcNAc, and aL-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with aGalNAc, GlcNAc, and L-Fuca1,2- Galb1,4GlcNAcb (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and nonbreeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

The present article reviews male camel behavior and breeding management strategies, providing an insight into the handling procedures and the most relevant welfare issues on these topics. Furthermore, it suggests some procedures for rearing, handling and collecting semen from camel bulls, based on results that have been achieved in the last twenty years and, recently, literature published with the aim of optimizing dromedary camel breeding. Camels are seasonal breeders and their breeding season (BS) is confined to the coolest winter months of the year; during the BS, also called “rutting period” or "rut", males exhibit morphological, behavioral and endocrinological peculiarities. Short breeding season, low libido and high aggressiveness are still some of the major cause of economic loss, poor reproductive performance and injuries, for camel breeding and industry. The application of ethology to approach, to train and to study camel bulls may be useful in the future to improve camel welfare and productive performances. Strong knowledge of animal learning and correct management procedure could be useful for camel technicians, owners, breeders, but also for veterinarians and others scientists.

Analyses of energy and redox status parameters are emerging technologies to improve oocyte quality assessment. Mitochondria (mt) play a vital role in the oocyte to support maturation, fertilization, and pre-implantation development. They are the major source of reactive oxygen species (ROS) produced during oxidative phosphorylation, which are not only by-products of cell metabolism but also important molecules for regulation of intracellular cell signaling. The aim of the present study was to test for mt/ROS colocalization in oocytes recovered from superovulated adult ewes and examined after in vivo or in vitro maturation (IVM). Cumulus–oocyte complexes of 8 superovulated (fluorogestone acetate þ D-cloprostenol for oestrus synchronization, pFSH/pLH and eCG for superovulation) adult (2 to 8 years of age) ewes were recovered (ovariohysterectomy by midventral laparotomy performed 54 h after vaginal sponge removal) either from flushing oviducts (oviducal oocytes) or from ovarian growing follicles (1–5 mm in diameter; follicular oocytes). Follicular oocytes were analysed after IVM (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mt, and ROS evaluation. Hoechst 33258 and Mitotracker Orange CMTM Ros were used to label nuclear chromatin and mt (Ambruosi et al. 2009) and 20,70-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353–360). Oocytes at the metaphase II (MII) stage showing regular ooplasmic size (4130 mm in diameter) and morphology were selected for confocal analysis of mt/ROS fluorescence distribution, intensity, and colocalization. Forty oviducal MII oocytes recovered from 8 ewes were analysed. Thirty-two oocytes recovered from the ovaries of 4 ewes underwent IVM, and 23 out of 32 (72%) reached nuclear maturation and were analysed. The rate of oocytes showing perinuclear mt distribution pattern did not differ between oviducal and IVM oocytes (33%, 13/40 v. 43%, 10/23; not significant). In these oocytes, fluorescent intensity of mt labelling and intracellular ROS levels did not differ between oviducal and IVM ooocytes (996.27 ` 363.57 v. 798.13 ` 275.91; not significant; and 1808.11 ` 442.78 v. 1473.29 ` 662.49, for mt and ROS, respectively; not significant), whereas mt/ROS colocalization was significantly higher in ovulated oocytes than in IVM oocytes (Pearson coefficient 0.67 ` 0.11 v. 0.39 ` 0.19, respectively; P o 0.001). In conclusion, in oocytes of adult ewes, mt aggregation, apparent energy status, and intracellular ROS levels do not differ between ovulated and IVM oocytes, but mt/ROS colocalization differs between the 2 groups. As it was reported for other cell systems that such a difference can be indicative of healthy status of ovulated oocytes, we suggest that mt/ROS colocalization could be considered as a suitable marker of oocyte quality.

This study describes regional differences in the oviduct of the one-humped camel (Camelus dromedarius) during the growth phase (GP) and the mature phase (MP) of the follicular wave by means of morphometry, scanning electron microscopy (SEM) and glycohistochemistry investigations. Epithelium height significantly increased in the ampulla and decreased in the isthmus passing from the GP to the MP. Under SEM, non-ciliated cells displayed apical blebs (secretory) or short microvilli. Cilia glycocalyx expressed glycans terminating with sialic acid linked 2,6 to Gal/GalNAc (SNA affinity) throughout the oviducts of GP and MP and sialic acid linked 2,3 to Gal1,3GalNAc (MAL II and KOH-sialidase (K-s)-PNA staining) throughout the MP oviducts. Non-ciliated cells displayed lectin-binding sites from the supra-nuclear cytoplasm to the luminal surface. Ampulla non-ciliated cells showed O-linked (mucin-type) sialoglycans (MAL II and K-s-PNA) during GP and MP and N-linked sialoglycans (SNA) during the MP. Isthmus non-ciliated cells expressed SNA reactivity in GP and MP, also K-s-PNA binders in MP, and MAL II and PNA affinity (Gal1,3GalNAc) during GP. Gal1,3GalNAc was sialilated in the non-ciliated cells of GP UTJ. Luminal surface lacked of Gal1,3GalNAc in GP and MP, whereas it expressed 2,6- and 2,3-linked sialic acids. In GP intraluminal substance reacted with SNA, MAL II, K-s-PNA in ampulla and only with MAL II in the isthmus and UTJ. These results demonstrate that the morphology and the glycan pattern of the camel oviductal epithelium vary during the follicular wave and that could relate to the region-specific functions.

OBJECTIVE: To analyze within-/between-subject, in vivo versus in vitro maturation (IVM), and age-related variations of mitochondrial (mt) bioenergy potential and oxidative status of metaphase II (MII) oocytes recovered from hormonally stimulated sheep. DESIGN: Prospective study. SETTING: Academic basic research laboratory. SUBJECT(S): Ten adult ewes. INTERVENTION(S):Estrus synchronization, controlled ovarian hyperstimulation (COH), ovariohysterectomy; follicular and oviductal oocyte retrieval; IVM of follicular oocytes. MAIN OUTCOME MEASURE(S): Mean ± SD, within-subject (CV(w)) and between-subject (CV(b)) variation coefficients of mt activity, intracellular reactive oxygen species (ROS) levels, and mt/ROS colocalization in sheep oocytes from young and aged donors and matured in vivo (in vivo MIIs) or in vitro (IVM MIIs). RESULT(S): Within- and between-subject, in vivo versus IVM, and age-related variations of mt activity were observed in MII oocytes from hormonally stimulated donor sheep. ROS levels increased significantly in oocytes from aged donors. Mt-ROS colocalization was consistently higher in in vivo MIIs compared with IVM MIIs. Oviductal energy/antioxidant ability is influenced by COH. CONCLUSION(S): Oocyte energy/oxidative status is affected by within-/between-subject, in vivo versus IVM, and age-related variations. Mt/ROS colocalization is a reliable marker of in vivo MII oocytes.

The study was carried out to monitor ovarian activity in female dromedary camels reared in semi-intensive system in Egypt, during non-breeding season, and to assess the effects of Controlled Intravaginal Drug Releaser (CIDR 1,38g) on vaginal environment and on follicular number and diameters. Twenty females were monitored through vaginal inspection and ultrasonographic examination. Group I (n=10) was monitored in July, while Group II (n=10) in September 2010. Follicle number and diameter were recorded before CIDR’s insertion (T0). In Group I CIDR’s were inserted after cleaning of perineum with dry paper while, in Group II, after washing of perineum and vaginal flushing with water-5% iodopovidone solution. After 10 days CIDR’s were removed, the vaginal status observed and follicles again counted and measured (T1). Results revealed that ovaries were active in July and even if in less measure, in September, which are considered non breeding season month in Egypt. CIDR’s treatment caused vaginitis in almost all Group I camels but a better vaginal environment. On the day 10, CIDR were removed in Group II. Statistical analysis revealed that the CIDR’s treatment significantly reduced mean follicular diameter in the two months (P<0.01; P<0.05 respectively) but did not affect follicular number, thus demonstrating its inefficacy in synchronize follicular wave in camels. Both ultrasonographic and hormonal studies will be necessary, simultaneously with CIDR treatment, for better understand effects of exogenous progesterone administration on ovarian activity and follicular wave pattern in female dromedary camels.

BACKGROUND: The sequencing of the cow genome was recently published (Btau_4.0 assembly). A second, alternate cow genome assembly (UMD2), based on the same raw sequence data, was also published. The two assemblies have been subsequently updated to Btau_4.2 and UMD3.1, respectively. RESULTS: We compared the Btau_4.2 and UMD3.1 alternate assemblies. Inconsistencies were grouped into three main categories: (i) DNA segments showing almost coincidental chromosomal mapping but discordant orientation (inversions); (ii) DNA segments showing a discordant map position along the same chromosome; and (iii) sequences present in one chromosomal assembly but absent in the corresponding chromosome of the other assembly. The latter category mainly consisted of large amounts of scaffolds that were unassigned in Btau_4.2 but successfully mapped in UMD3.1. We sampled 70 inconsistencies and identified appropriate cow BACs for each of them. These clones were then utilized in FISH experiments on cow metaphase or interphase nuclei in order to disambiguate the discrepancies. In almost all instances the FISH results agreed with the UMD3.1 assembly. Occasionally, however, the mapping data of both assemblies were discordant with the FISH results. CONCLUSIONS: Our work demonstrates how FISH, which is assembly independent, can be efficiently used to solve assembly problems frequently encountered using the shotgun approach.

Generally cervical mucus penetration test (CMPT) have been used for assessment of semen quality. In this study, a simplified CMPT was applied to evaluate quality of cervical mucus of cows in estrus using semen of proven fertility. Viscosity, pH, spinnbarkeit, cellularity, crystallization and penetration, were measured. A significant positive linear correlation was found between sperm concentration [Sp] detected in mucus after CMPT and the quality of each mucus samples according to physical characteristics. Comparing [Sp] values of cyclic cows with those of pathologic ones, we obtained higher [Sp] for cyclic cows respect to the pathologic cows. The evaluation of [Sp] could be useful to improve the diagnostic framework of bovine fertility.

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955-963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142-1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P<0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P<0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P<0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n=29) and control (n=36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2',7'-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720-728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8±499.5 v. 722.9±390.3; DCF fluorescence intensity, 278.5±179.3 v. 378±185, and mt/ROS colocalization, 0.5±0.1 v. 0.5±0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.

ABSTRACT - The purpose of this review article is to present a 50-year perspective of research on mechanical method for limiting male dog reproduction by the use of therapeutic ultrasound. Ultrasound's potential as a male contraceptive was first reported by Fahim et al. in 1977 where it was shown that a single application could result in a reversible dramatic loss of germ cells. If the method can be made permanent, a noninvasive method for controlling domestic pet populations could be developed, although standard treatment is not yet identified. More recent studies in 2000s by our research group demonstrated that tree treatment

An ideal contraceptive for male dogs should be 100% efficient, irreversible, inexpensive and with no side effects. Ultrasound was used as a male contraceptive in several species, including dogs. Studies are needed to determine minimal number of treatments, interval between treatments, part of testes that requires treatment, frequency or power of ultrasonic wave exposure and many more need to be investigated before its application in practice. A previous study concluded that three treatments of 5 min/treatment per day (2.5 cm2 transducer, 1 MHz, 1.5 W/cm2) are ineffective. In this trial, effects of two testicular ultrasound exposure protocols (differing in ultrasound treatment length and number of applications) on testicular size, consistency and volume, and sperm concentration and motility were evaluated. Twenty dogs were divided in two equal groups- A and B. All subjects were exposed to 1.5 Wcm2 of ultrasound on each testicle using Vetrison Portable ultrasound (Physiomed Elektromedizin AG, Germany; 2.5 cm2 transducer). Dogs in group-A received ultrasound for 5 min on alternate days for one week and dogs in group-B received ultrasound for 15 min inutes twice a day on alternate days. Sperm concentrations and motility evaluations were made before and 25 days after the end of treatments. Length and testicular width were echographically measured to calculate the volume. Semen collected was examined by using an integrated visual optical system for semen analysis for sperm concentration and for percentage of total and progressively motile sperms. All dogs were castrated at day 40 and gonads were collected for histological examination. Data concerning testicular volume were statistically analyzed with ‘Wilcoxon matched pairs signed rank sum’ test (p £ 0.05); semen evaluation was statistically analyzed with ANOVA test (p £ 0.01). After ultrasound treatment, all dogs showed no local or systemic adverse effects, and no pain or skin burns. However, dogs in group A exhibited marked tenderness of testicles at palpation. Group A dogs showed a statistically significant reduction of the volume of both testis (left 9.6 ± 3.7 vs. 5.5 ± 3.6/cm3; right 9.6 ± 2.7 vs. 3.6 ± 1.4/cm3; p £ 0.05) while no reduction in testicular volume was noticed in dogs in group (left: 9.1 ± 1.4 vs. 9.2 ± 1.6/cm3; right: 9.3 ± 1.5 vs. 9.4 ± 1.7/cm3). Before the US treatment, mean volume of ejaculates was 10 ± 3.5 ml, sperm concentration was 300.8 ± 24.8 · 106/ml with an average percentage of total and progressive motile sperms of 88.2 ± 4.5 and 59.3 ± 5.3, respectively. After the ultrasound treatment, a zero sperm count was noticed in group A dogs (p £ 0.01), and no variation in B group. Histology evaluation showed interstitial fibrosis, widespread tubular atrophy and hyalinization of the basement membranes in group-A dogs and no changes were observed in group-B dogs. Our results demonstrated that ultrasound treatment for 5 min on alternate days for one week leads to irreversible testis damage consistent with permanent sterilization, while reducing the number of applicationseven with a longer treatment was ineffective on dog fertility

The camel (Camelus dromedarius) represents an important economic resource in many arid areas across several countries, as a pack or racing animal or since providing milk, meat and hair. Despite its relevance, an exhaustive survey of the genetic variability among camel populations in North Africa is still lacking due to absence of cross-border studies in the currently available literature. As a consequence, a collaborative effort was recently launched to fill this gap. The project is now in its infancy stage. Here we describe the genesis of the project and provide preliminary details on the methodological approach. Up to now, 310 blood samples, representative of the different camel types found in Tunisia, together with 214 blood samples from Algeria and 78 blood samples representative of the north western coast of Egypt have been sampled. Further efforts are currently being made in order to ensure a good representativeness for camel populations in Egypt. A set of 20 FAO STR has been adopted. Analysis of data is expected to provide original insights on the historical process of camel dispersal in Northern Africa and contribute to a better characterization of camel populations in this region.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 x 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 x 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.

BACKGROUND: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. METHODS: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. RESULTS: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts. CONCLUSIONS: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.