Induction of sexual activity in male and female farmed ostriches (Struthio camelus) with GnRH implant

The annual photoperiodic changes are the most important factor controlling the reproductive activity in birds. A single subcutaneous implant of a slow-release GnRH analogue induced the seasonal reproductive activity in both sex of farmed ostriches. Significant increase in annual egg production and clutch number compared with control group were observed

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

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

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

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>Our work demonstrates how FISH, which is assembly independent, can be efficiently used to solve assembly problems frequently encountered using the shotgun approach.</p

Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

The present study investigates the effects of high external calcium concentration ([Ca(2+)](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.A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](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 [Ca(2+)](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.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 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 (&gt; 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. © 2021 The Authors. Veterinary Medicine and Science published by John Wiley &amp; Sons Ltd.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 (&gt; 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