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One of the main drawbacks of using endometrial cytology in cows is the time required for sample collection and interpretation. It is recommended to count a large number of polymorphonuclear neutrophils (PMN) and to calculate their overall percentage. However, since counting a large number of cells is a laborious method, it would be preferable to simplify the analysis by counting the number of PMN in few microscopic fields. Therefore, the aim of this study was to assess whether a simple test, based on calculating the average number of PMN in 10 fields at 1000x, could be a reliable technique for the diagnosis of endometritis. Two hundred and sixty endometrial samples were taken from Holstein cows at different postpartum stages using an adapted cytobrush. Smears obtained were air-dried for fixing and stained with a Romanowsky-type procedure. To evaluate the counting method, the percentage of PMN in 150 cells was calculated as well as the average number of PMN in 10 fields at 1000x. Receiver operating characteristic (ROC) curves was constructed to evaluate both methods, the percentage of PMN (used as reference) and the average number of PMN. It was observed that the area under the curve is (regardless of cut-of used) higher than 0.99 and the correspondence between both methods were 1.58 PMN/field for the cut-of value of 15% and 2.40 PMN/field for the cut-of value of 20%. These results show that this simple method could be used to determine the percentage of PMN in endometrial cytological samples and to diagnose endometritis in cows. [ABSTRACT FROM AUTHOR]

The objectives of the study were to evaluate the ovarian follicular response, cumulus–oocyte complex (COC) collection rate, fertilization, and culture of COC collected from alpacas after treatment with 2 different gonadotropins. Female alpacas were assigned to Group 1 (n=8), 200mg of FSH (Folltropin, Bioniche, Belleville, Ontario, Canada) divided b.i.d. for 3 days, plus a single IM dose of 1000 IU of hCG (Chorulon, Intervet, Salamanca, Spain) 24h after the last FSH treatment; or Group 2 (n=10), 750 IU of eCG (Folligon, Intervet) as a single dose, plus a single IM dose of 1000 IU of hCG on Day 3 after eCG treatment (Day 0=start of the superstimulatory treatment). At 20 to 22h post-hCG treatment, the ovaries were surgically exposed and COC were aspirated from follicles ≥6mm and evaluated. The COC with a homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of TCM-199 maturation medium supplemented with 10% FCS (vol/vol) plus 0.5μgmL-1of FSH, 10μgmL-1of hCG, 0.2mM sodium pyruvate, 50μgmL-1of gentamicin, and 1μgmL-1of oestradiol under mineral oil with 10 to 12 oocytes/drop and maturated 24h at 39°C in an atmosphere of 5% CO2and high humidity. After maturation, COC were removed and fertilized in vitrousing epididymal sperm. Testes were collected from mature males from a slaughterhouse and transported to the laboratory. The caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation at 600×gon a Percoll discontinuous gradient (45.0:22.5%) for 10min. The supernatant was then removed by aspiration and the pellet was resuspended in TL-HEPES and centrifuged again at 300×gfor 5min. The pellet was resuspended in TL-stock. Gametes were co-incubated for 18h at 39°C with 5% CO2and high humidity. Presumptive zygotes were cultured in KSOM medium supplemented with 1mM glutamine, 0.3mM sodium pyruvate, 50μgmL-1of gentamicin, EDTA, essential and nonessential amino acids, and BSA for 3 days and cultured in SOF medium for 7 days. Embryo development was evaluated at 72h and 7 days. Data were subjected to ANOVA. The number of follicles ≥6mm did not differ at the time of COC collection (19.3±5.7 and 21.5±7.3), and the number of COC collected was 16.7±5.3 and 17.3±6.6 for the FSH group and the eCG group, respectively. The cleavage rate was 45.2 and 42.1% for the FSH group and the eCG group, respectively, at 72h of culture, and the blastocyst stage at Day 7 (22.2 v.19.3) did not differ between treatments. In conclusion, the FSH and eCG treatments did not differ in the ovarian follicular response, COC collection rate, fertilization, and culture of COC. Both gonadotropins can be used in the IVF protocol for alpacas. Grant 064 FINCyT-PIBAP 2008 and Grant 032-2009 PROCYT–CONCYTEC. [ABSTRACT FROM AUTHOR]

We have previously reported that alpaca oocytes require between 38 and 42h of maturation time (Huanca et al.2010 Reprod. Fertil Dev. 22(1), 327). The objective of this study was to evaluate the effect of the addition of FSH in the maturation medium on nuclear maturation and cleavage rate. Ovaries were collected from a slaughterhouse and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. Cumulus–oocyte complexes (COC) were obtained by slicing of ovaries with a scalpel and were pooled in a conical tube for sedimentation before evaluation. The 476 COC with homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μmL drop of maturation medium TCM-199 supplemented with 10% FCS (vol:vol), 10μgmL-1of hCG, 0.2mM sodium pyruvate, 50μgmL-1of gentamicine, and 1μgmL-1of oestradiol plus 0.5μgmL-1of FSH (Folltropin, Bioniche Animal Health, Belleville, Ontario, Canada) according the following treatments: T1 (Control): FSH by 42h, T2: 21h with FSH+21h without FSH, T3: 21h without FSH+21h with FSH. The COC were maintenance under mineral oil with 10 to 12 oocytes per drop and maturated 42h at 39°C in an atmosphere of 5% CO2and high humidity. After the maturation time, part of the COC were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1mg/mL of hyaluronidase and fixed in ethanol: acetic acid (3:1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5min. The slides were examined under a phase contrast microscope at ×400 to evaluate the status of nuclear maturation and classify as germinal vesicle (GV), metaphase I (MI), anaphase–telophase, metaphase II (MII), and degenerated. The other part of oocytes was fertilized in vitrousing epididymal sperm. Motile spermatozoa were obtained by centrifugation at 600×gon a percoll discontinuous gradient (22.5:45.0%) for 10min. After the supernatant was removed by aspiration, the pellet was resuspended in TL HEPES and centrifuged again at 300×gby 5m. The pellet was resuspended in TL Stock. Gametes were coincubated for 18h at 39°C with 5% CO2and high humidity. Presumptive zygotes were culture in KSOM supplemented with 1mM of glutamine, 0.3mM of sodium pyruvate, 50μgmL-1of gentamicine, ethylenediaminetetraacetic acid, amino acids essentials and nonessentials, and BSA by 3 days and cultured in SOF medium for 7 days. Cleavage rate was evaluated at 72h. Proportional data were compared by chi-square test. The proportions of oocytes reaching MII stage were 64.9±8.1, 49.2±9.4, and 53.8±7.5% for T1, T2, and T3, respectively. Cleavage rates were 39.1, 36.3, and 33.1% and blastocysts rates were 13.6, 16.1, and 14.5% for T1, T2, and T3, respectively. The results suggest that the moment of addition of FSH does not have an effect on the maturation and cleavage rate of alpaca oocytes. This work was supported by Grant 064 – FINCyT – PIBAP 2008 and Grant 032 – PROCYT – CONCYTEC. [ABSTRACT FROM AUTHOR]

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1FSH, 10 μg mL-1hCG, 0.2 mMsodium pyruvate, 50 μg mL-1gentamicine, and 1 μg mL-1Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mMsodium pyruvate, and 50 μg mL-1gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P< 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008. [ABSTRACT FROM AUTHOR]