Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pick up (OPU) would induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. In this work, we compared three protocols for OPU and in vitro production (IVP) of embryos, in Nellore cattle. Nellore cows (n = 18) were randomly allocated in three groups: Group 1 (OPU), Group 2 [Follicle stimulating hormone (FSH) and OPU] and Group 3 (FSH deprivation and OPU). Three OPUs were performed, and the animals were switched to a different group each time (crossover), in such a way that at the end of the experiment all cows received the 3 protocols. At random stage of the oestrous cycle (D‐2), all follicles ≥ 6 mm were aspirated to induce a new follicular wave 2 days afterwards (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superstimulated (FSH, Folltropin^®, 30 mg administered daily, i.m., during three consecutive days, total dose = 180 mg), and 6 h after the last FSH dose, they received exogenous luteinizing hormone (LH) (12.5 mg, i.m., Lutropin^®, D3). The OPU was performed 6 h after LH administration, i.e. 12 h after the last dose of FSH. Animals in Group 3 received the same treatment as those in Group 2, except that LH was administered 42 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this group, follicles were deprived of FSH at 48 h. Both cleavage and blastocyst rates were similar (p > 0.05, anova ) among oocytes from Groups 1, 2 and 3, respectively: 77.4% (144/185) and 42.70% (79/185); 75.54% (105/139) and 31.65% (44/139); 63.52% (101/159) and 33.33% (53/159). However, hatched blastocyst rate was higher (p < 0.01) in Group 1 (30.27%, 56/185) when compared with Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (Bos taurus), ovarian superstimulation associated with deprivation of FSH and OPU (Group 3) did not increase IVP of Nellore embryos (Bos indicus). On the contrary, the highest hatched blastocyst rates were observed in oocytes from non‐superstimulated cows.     

Endotracheal intubation in horses: a study of two cuff inflation pressures, correlation with liquid aspiration, and tracheal wall damage

Nine adult horses were anesthetized for a nonsurvival abdominal adhesion study. Horses were randomly assigned into two groups to receive endotracheal tube cuff pressures of either 80 cm H₂O (Group P80) or 120 cm H₂O (Group P120). After intubation (Bivona 30 mm ID), anesthesia was maintained with isoflurane. Horses were ventilated 10 times per minute with a suitable inspiratory pressure to maintain Pe ′CO₂ in the 35–40 mm Hg (4.7–6.0 kPa) range. Cuff pressure was continuously monitored with a pressure transducer (TruWave, Baxter) calibrated to the atmospheric pressure and maintained at a constant pressure. Twenty‐five millilitres of methylene blue dye in saline were instilled proximal to the cuff over 5 minutes. The horses were euthanized 123 ± 23 minutes later (mean ± SD). Immediately, the trachea was opened distal to the tip of the endotracheal tube, and the mucosa was observed for evidence of dye leaking past the cuff. The cervical trachea was resected and the lumen exposed by a ventral longitudinal incision. Biopsies (1–2 rings) were obtained at mid‐cuff level and distal to the tip of the endotracheal tube, and placed in formalin for later histologic examinations (H&E stain). Methylene blue stain was not observed distal to the endotracheal tube cuff in any horse. Visual examination of the tracheal mucosa revealed hyperemic or hemorrhagic lesions at the level of cuff contact both ventrally and dorsally. Histologic changes included epithelium damage, submucosal neutrophil infiltrates, and acute submucosal hemorrhages. P80 horses had none or focal to multifocal lesions on the ventral and dorsal aspects of the rings. P120 horses had multifocal to diffuse lesions on all aspects (dorsal, ventral, and lateral). We concluded that the endotracheal tube cuff produced a seal sufficient to prevent leakage at both pressures. Tracheal damages on gross and microscopic examinations were more severe and occurred more frequently at the higher cuff pressure.     

Seasonal Changes in Testicular Cell Morphology in Domestic Male Cats (Felis catus)

The aim of this study was to asses the variation in the morphology of the seminal epithelium in relation to natural photoperiod in male cats. Tom cats (n = 240) were castrated every other week throughout the year. Each testis was fixed in Bouin's solution and cut into sections. The percentage of tubules with round spermatids (RS), elongated spermatids (ES), tailed spermatids (TS), mature spermatids (MS) and the number of Sertoli cells (SC) and Leydig cells (LC) were recorded in each sample. Testicles from males during short days (SHD) had a higher percentage of tubules with RS and ES compared to testicles from males during long days (LHD, 31.3 ± 0.6 vs 2.1 ± 0.6%, p < 0.001; 30.9 ± 0.7 vs 11.0 ± 0.7%, p < 0.001). Conversely, testicles from males during SHD had a lower percentage of tubules with TS and MS compared to testicles from males during LHD (24.5 ± 0.8 vs 29.7 ± 0.8%, p < 0.01; 13.1 ± 1.2 vs 57.0 ± 1.2%, p < 0.01). Furthermore, testicles from males during SHD had a higher number of SC and lower number of LC compared to testicles from males during LHD (11.4 ± 0.1 vs 8.0 ± 0.1%, p < 0.01; 19.2 ± 1.0 vs 38.0 ± 1.0%, p < 0.01). In conclusion, there are seasonal changes in testis cell morphology in the tom which may be related to seasonal sperm production.     

Cryopreservation of Sheep Primordial Follicles

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.     

Resident forum abstractsEVALUATION OF THROMBOELASTOGRAPHY (TEG) IN NORMAL CATS

Objective: To establish normal parameters of thromboelastography (TEG) in healthy adult cats. Background: Thromboelastography (TEG) is an in vitro test of coagulation that has been shown to be useful in humans, dogs and select species to identify and quantify alterations of hemostasis (e.g., hypercoagulable and hypocoagulable states). It has also been demonstrated to be useful in monitoring effects of anticoagulant therapies. This test has not been evaluated in cats. Methods: Blood was collected from 25 clinically normal cats by venipuncture using a 21 gauge×3 1/2 inch butterfly catheter and syringe for medial saphenous or jugular venipuncture. A single 1.8 mL sample in 3.8% Sodium Citrate (9:1) was collected from each cat. Recalcified whole blood was analyzed 30 minutes following collection with the TEG® 5000 analyzer (Haemoscope, Niles, IL). Analysis temperature was 37.6°C. TEG parameters recorded included: R‐value (represents initial fibrin formation), K (time from R to standard fixed measure of clot firmness which represents contributions of platelets and fibrinogen), maximum amplitude (MA; represents absolute clot strength), and alpha angle (α; the slope of TEG tracing which represents rate of clot formation). The coagulation index (CI) was derived from the formula generated for humans to provide an overall assessment of whether the sample was hyper‐ or hypocoagulable. Results: Values for the 25 normal cat samples are reported as mean ±2 standard deviations. R=2.97; 1.23–4.72; K=1.54, 0.38–2.71; α=70.70, 57.76–83.65; MA=58.50, 45.26–71.74 and CI=2.27, 0.07–4.46. Compared to historical information obtained on normal dogs, cats have significantly shorter R and K and larger α, MA and CI. Conclusions: TEG does have reproducible performance when used to evaluate coagulation status in normal cats. Compared to dogs, normal cats favor a hypercoagulable state. Species‐specific normal values are necessary for interpretation of TEG results. This test bears potential value for use in future experimental and clinical work to investigate hemostasis in cats receiving anticoagulant therapies or in cats suffering from diseases such as cardiomyopathy which are thought to be associated with altered coagulation status.