Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Post-thawing effects of three cryopreservation diluents on Rusa deer (Rusa timorensis) spermatozoa.

The aim of this study was to evaluate home-made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES-based, Tris-based and Triladyl® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post-thawing and after 1-hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post-thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl® and TES (p < 0.05) as compared with Tris (PM of Triladyl® 14.7% vs. 3.2% TES and 2.5% Tris; VSL 56 for Triladyl® , 59.2 for TES and 41.7 for Tris; LIN 45.6 for Triladyl® , 52 for TES and 36.5 for Tris). Triladyl® and TES extender led to better post-thawing sperm parameters, but these preliminary results need to be verified through artificial insemination trials.

Ejaculation effect on blood testosterone and prostatic pulsed-wave Doppler ultrasound in dogs.

Local vessels ultrasonography evaluates prostatic physio-pathologic states. Testosterone promotes tissue and vascular growth. Knowing variables on prostatic vasculature is crucial to correctly apply Pulsed-Wave exam. The study aims to assess how ejaculation and blood testosterone affect Pulsed-Wave indexes. Serial blood testosterone dosages and Pulsed-Wave exams were performed in 20 dogs, immediately before (T0) and after (T1) ejaculation and 6 hr later (T2). Arteria prostatica cranialis, Arteriola capsularis, Arteriola trabecularis and Arteriola parenchimalis were evaluated and mean Pulsatility and Resistivity Index, Systolic-Peak, End-Diastolic and Mean Velocity calculated. Data were grouped by time and vessel (ANOVA, p ≤ 0.05). At T1, Resistivity Index significantly lowered in A. prostatica cranialis, A. trabecularis and A. parenchimalis but grew in A. capsularis; Pulsatility Index had the same pattern, but not significant in A. parenchimalis; Systolic Peak Velocity, End-Diastolic Velocity, Mean Velocity significantly rose in A. capsularis and A. trabecularis. No indexes differed at T0 and T2. Testosterone did not differ at T0 (10.93 ± 7.05 ng/ml), T1 (12.71 ± 7.29) and T2 (10.54 ± 6.63). Results stated the risen prostatic vascular flow postejaculation, affecting Pulsed-Wave. Due to semi-rigid capsule, impairing vasodilation of other vessels, only A. capsularis indexes increased. Intimal cushions of A. prostatica cranialis kept velocities fixed; A. capsularis and A. trabecularis lack of intimal cushions, thus velocities grew. In A. parenchimalis, precapillary sphincters opening allows increased flow redistribution in vasodilated parenchymal bed, keeping velocities fixed. As testosterone, not affected by ejaculation, did not peak, vascular changes are not due to testosterone itself. These physiological effects of ejaculation suggest proper sexual rest before Pulsed-Wave exam planned to explore suspected prostatic neovascularization.

Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

BACKGROUND: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts. METHODS: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups. RESULTS: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status. CONCLUSIONS: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

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.

Confocal fluorescence assessment of bioenergy/redox status of dromedary camel (Camelus dromedarius) oocytes before and after in vitro maturation.

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.

Vitrification preserves chromatin integrity, bioenergy potential and oxidative parameters in mouse embryos.

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.

Tissue sealing versus suture ligation in open canine ovariectomy: Surgical times, intraoperative nociceptive response and frequency of complications.

BACKGROUND: In this study, we compared two different techniques currently used for open canine ovariectomy: traditional method utilising absorbable suture and vessel sealing device (ENSEAL® Ethicon Endo-Surgery, Cincinnati, OH). OBJECTIVES: The aim of this study was to compare the surgical times, intraoperative nociceptive response and the frequency of intraoperative complications in the canine ovariectomy procedure using these two techniques. METHODS: Forty bitches were randomly divided into two groups. The Control Group (C) will use a classic open surgery approach using ligatures with absorbable suture and ovarian resection with a scalpel blade. In the Group E, resection of ovarian structures was performed with ENSEAL® tissue sealer device. For each dog the surgical times, the intraoperative nociceptive response (measuring heart rate, respiratory rate and non-invasive blood pressure) and the intraoperative complications were measured to compare the effectiveness of the two techniques. RESULTS: The results of this study showed that the procedures performed using ENSEAL® were faster than the traditional techniques using surgical suture. Instead, the results regarding the nociception and the safety of the two procedures are similar. CONCLUSIONS: The present study shows that the use of ENSEAL® significantly shortened the surgical time. Meanwhile, its use was found to be similarly safe and efficient in terms of intra-operative nociception, as the classical techniques with absorbable suture. Canine ovariectomy using ENSEAL® device is more practical and faster than the traditional technique; the routine use of this device is considered a useful alternative for the canine neutering.

The effect of splash block on the need for analgesia in dogs subjected to video-assisted ovariectomy.

OBJECTIVES: The present study aimed to demonstrate the efficacy of splash block using lidocaine to provide additional analgesia during ovariectomy in bitches. To identify an acute intraoperative nociceptive response, three clinical parameters were used: increased blood pressure, heart rate and respiratory rate. MATERIAL AND METHODS: Forty healthy bitches were randomly assigned to receive 2% lidocaine (L group) topical application (splash block) on both ovaries (2 mg/kg each), or an equal volume of NaCl 0.9% at the same sites (C group). A fentanyl bolus (2 µg/kg) was administered intraoperatively in response to an increase in blood pressure, heart rate or respiratory rate during surgery (> 30% compared with the pre-incisional values). RESULTS: Local lidocaine administration significantly reduced the need for supplementary fentanyl. Dogs in the L group showed greater intraoperative hemodynamic stability and lower surgical pain than those in the C group. In addition to the routine anaesthetic protocol, the local anaesthesia used in the present study was safe and caused no cardiopulmonary suppression. In addition, it significantly reduced the need for mandatory systemic or rescue analgesia. CLINICAL SIGNIFICANCE: Ovariectomy is a common surgical procedure in bitches. Analgesia during this procedure is important because intraoperative pain can cause negative effects that prevent patient recovery. This study aimed to demonstrate the analgesic efficacy of lidocaine splash block in video-assisted ovariectomy in dogs. The results showed that splash block improved surgical analgesia during canine laparoscopic ovariectomy. Considering its relative simplicity, low cost, and safety, splash block could be used in daily clinical practice.

Fentanyl patch versus tramadol for the control of postoperative pain in canine ovariectomy and mastectomy.

OBJECTIVES: The purpose of the present study was to evaluate the postoperative analgesic efficacy of fentanyl patches versus subcutaneous tramadol after canine ovariectomy, with and without unilateral mastectomy. MATERIALS AND METHODS: A total of 40 female dogs were included in the present study, all of which were domesticated, healthy and 4-12 years of age. The animals were divided into four groups (n = 10 per group) based on the surgery and the analgesic protocol used: the TO group only underwent ovariectomy, and received postoperative tramadol; the TM group underwent both ovariectomy and mastectomy, and received postoperative tramadol; the FO group only underwent ovariectomy, and received fentanyl patches; and the FM group underwent both ovariectomy and mastectomy, and received fentanyl patches. Postoperative pain was evaluated every 4 h for 24 h using a numeric analogue scale (NAS) and a modified Glasgow Composite Measure Pain Scale Short Form (CMPS-SF). RESULTS: The results of the present study showed that patients in all four groups tolerated postoperative surgical stress well. Analysis of variance for repeated measures did not show significant differences in the NAS scores and in Glasgow CMPS-SF between groups in terms of pain scores or rescue analgesia. CLINICAL SIGNIFICANCE: These results indicated that the analgesic effect of the fentanyl patch was similar to that of subcutaneous (SC) tramadol in female dogs after ovariectomy, with and without unilateral mastectomy, suggesting that the fentanyl patch may represent a valid supplementary tool for the control of postoperative pain in animals after surgery.

Local and Regional Anaesthetic Techniques in Canine Ovariectomy: A Review of the Literature and Technique Description.

Canine ovariectomy is an elective surgery with a moderate level of pain. Despite its relative simplicity, it requires surgical pain management. This study aimed to collect all recent information about local and regional anaesthetic/analgesic techniques in a review of the literature describing the technique utilised. The various procedures described in this review use local anaesthetics to improve analgesia in the routine systemic anaesthetic protocol. The approach described in this paper is called multimodal analgesia and is used in addition to the normal standard anaesthetic protocol. These techniques proved effective in minimising responses to the surgical stimulus and ensured adequate intraoperative and postoperative analgesia. The routine use of multimodal analgesia is considered a useful alternative for pain management in canine ovariectomy, in that it minimises patient suffering, improves the recovery of rescue analgesia, increases drug savings, and improves animal outcomes. In addition, the use of these local and regional techniques ensures satisfactory analgesic coverage that lasts for the first hours postoperatively.

Refinement of Bos taurus sequence assembly based on BAC-FISH experiments.

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.

Ultrasound-Guided Funicular Block: Ropivacaine Injection into the Tissue around the Spermatic Cord to Improve Analgesia during Orchiectomy in Dogs.

Orchiectomy is a common surgical procedure performed on small animals, and it requires postoperative pain management despite its relative simplicity. This study aimed to evaluate the hemodynamic stability, intraoperative administration of additional hypnotic and/or analgesic drugs, and postoperative pain scores following the combination of ultrasound-guided injection of ropivacaine hydrochloride into the spermatic cord and infiltration by the same anaesthetic of the incisional prescrotal line (ROP) or general anaesthesia. Dogs in the ROP group showed greater intraoperative hemodynamic stability and lower pain scores than the control group. The locoregional approach used in this study proved effective in minimising the responses to the surgical stimulus and ensured adequate analgesia intra- and postoperatively. This method, called ultrasound-guided funicular block, allows orchiectomy to be performed under deep sedation without general anaesthesia.

Use of Spinal Anaesthesia with Anaesthetic Block of Intercostal Nerves Compared to a Continuous Infusion of Sufentanyl to Improve Analgesia in Cats Undergoing Unilateral Mastectomy.

Unilateral mastectomy is a common surgical procedure in feline species and requires postoperative pain management. Our study aimed to evaluate the analgesic efficacy of subarachnoid anaesthesia combined with an intercostal nerve block, in comparison with the use of sufentanyl citrate administered as a constant-rate infusion (CRI). Twenty cats were randomly divided into two groups (n = 10/group) based on the analgesic protocol used: the first received loco-regional anaesthesia with levobupivacaine (LR group), and the second received a CRI of sufentanyl (SUF group). The evaluation criteria during surgery were the need for a bolus of fentanyl in the event of an increased heart rate or increased blood pressure. In the postoperative period, the levels of comfort/discomfort and pain were used to obtain a score according to the UNESP-Botucatu multimodal scale. Subjects who scored above seven received analgesic drug supplementation. Intraoperative analgesia was satisfactory, with good haemodynamic stability in both groups. Four patients in the LR group required an extra dose of methadone after they achieved the sternal decubitus position, whereas those in the SUF group required many more doses. The analgesia achieved in the LR group was more satisfactory than that in the SUF group.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ejaculation Effect on Canine Prostatic Specific Esterase Serum Concentration.

Canine prostatic diseases are usually asymptomatic in their onset and often identified in advanced stages. Canine prostatic specific esterase (CPSE) represents an early serum marker for prostatic diseases, also in asymptomatic dogs. The present study aimed to identify the effects of ejaculation on serum CPSE. Twenty adult intact male dogs were enrolled. Blood samples were collected to measure CPSE concentrations before (T0), immediately after (T1), and 24 h post (T2) ejaculation. Data were compared within and between groups by ANOVA (p < 0.05). Dogs were divided in two equal groups: A (healthy: CPSE ≤ 52.3 ng/mL at T0) and B (suspected for prostatic disorders: CPSE > 52.3 ng/mL or diagnosed with symptoms of prostatic diseases: CPSE > 90 ng/mL). CPSE was shown to be statistically higher in group B than A at any time point. In both groups, CPSE showed a significant increase at T1, and no significant differences between T0 and T2. This study demonstrates a definite effect of ejaculation on CPSE concentration. Twenty-four hours post-ejaculation, CPSE returns to basal values. Such physiological effects of ejaculation should be considered when planning analyses of CPSE concentrations, i.e., by respecting a proper sexual rest.

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse.

BACKGROUND: The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI). METHODS: Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining. RESULTS: In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere. CONCLUSION: Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin.

The Effect of Dietary Supplementation of Vitamin E, Selenium, Zinc, Folic Acid, and N-3 Polyunsaturated Fatty Acids on Sperm Motility and Membrane Properties in Dogs.

Sub-fertility represents a common challenge in canine reproduction. Different protocols, supplementing daily given quantities of micronutrients, were investigated to improve poor sperm concentration and/or function, which represent breeding major constraining factors in the canine species. Little information is available for dogs concerning the effect of a daily supplementation with a complex of vitamin E, zinc, selenium, folic acid, and n-3 polyunsaturated fatty acids (PUFA) on semen quality. Thus, the present study investigated this effect on semen motility and sperm membrane properties. Serial semen analyses from fourteen healthy normospermic dogs, fed with the same commercial diet, were performed on Days 0 (T0), 30 (T30), 60 (T60), and 90 (T90). Seven dogs were randomly included in the treatment (T) group, receiving a supplementation of vitamin E, zinc, selenium, folic acid, n-3 PUFA; and seven other subjects composed the control (C) group. Total Sperm Count (TSC), Computer-Assisted Sperm Analysis (CASA) indexes, mortality, and functional membrane integrity were assessed. The ANOVA compared results between groups and sampling times (p < 0.05). From T60, the supplementation significantly improved TSC, progressive motility, functional membrane integrity, and decreased mortality. Present results lead us to consider ameliorative effects of a two-month healthy diet supplementation on canine spermatozoa. The positive effects of the described balanced integration of micronutrients on sperm motility and prevention of oxidative stress should be considered, especially when decreased seminal parameters may result from inadequate intake, reduced absorption, increased losses or demand, or to attenuate the impact of age.

Alcohol diluent provides the optimal formulation for calcium chloride non-surgical sterilization in dogs.

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.

A dose-finding, long-term study on the use of calcium chloride in saline solution as a method of nonsurgical sterilization in dogs: evaluation of the most effective concentration with the lowest risk.

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.