Feline ovarian tissue vitrification: The effect of fragment size and base medium on follicular viability and morphology.

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.

Assessment of the temperature cut-off point by a commercial intravaginal device to predict parturition in Piedmontese beef cows.

Dystocic parturitions have an adverse impact on animal productivity and therefore the profitability of the farm. In this regard, accurate prediction of calving is essential since it allows for efficient and prompt assistance of the dam and the calf. Numerous approaches to predict parturition have been studied, among these, measurement of intravaginal temperature (IVT) is the most effective method at the field level. Thus, objectives of this experiment were, 1) to find an IVT cut-off to predict calving within 24 h, and 2) to clarify the use of IVT as an automated method of calving detection in housed beef cows. A commercial intravaginal electronic device (Medria Vel'Phone®) with a sensor that measures the IVT every 12 h was used. Piedmontese cows (n = 211; 27 primiparous and 184 multiparous) were included in this study. One-way analysis of variance was used to assess the temperature differences at 0, 12, 24, 36, 48 and 60 h before parturition. Receiving operator characteristic curves were built to determine the temperature cut-off which predicts calving within 24 h with the highest summation of sensitivity (Se) and specificity (Sp). Binomial logistic regression models were computed to identify factors that may affect the IVT before calving. Mean gestation length was 291.5 ±â€¯13.7 d (primiparous, 292 ±â€¯14.1 d; multiparous, 289 ±â€¯9.2 d). A decrease (P < 0.001) in the average IVT was found from 60 h before calving until the expulsion of the IVT device. A significant (P < 0.05) reduction in the IVT was noticeable from 24 h before until parturition. The IVT drop to predict parturition 24 h before calving was 0.21 °C (area under the curve [AUC] = 0.72; Se = 66%, Sp = 76%). Furthermore, the IVT cut-off value to predict parturition within 24 h was 38.2 °C (AUC = 0.89; Se = 86%, Sp = 91%). None of the evaluated fixed effects (parity, dystocia, season or length of gestation) affected (P ˃ 0.05) the IVT variation from 60 h before and up to calving. To conclude, the IVT average seems to be a better parameter than the drop in temperature to predict parturition within 24 h. In this regard, a cut-off of 38.2 °C showed a high Se and Sp for predicting calving. This study demonstrates the usefulness of a commercially available device to predict calving to improve management in stabled beef farms.

A novel cytologic sampling technique to diagnose subclinical endometritis and comparison of staining methods for endometrial cytology samples in dairy cows.

The present article describes a study of the diagnosis of subclinical endometritis in dairy cows having two principal aims: first, to validate a novel technique for taking endometrial cytology samples to diagnose subclinical endometritis in dairy cows. Second, to compare the percentage of polymorphonuclear cells (PMNs) in cytology samples stained with Diff-Quik versus a specific staining method for PMNs, naphthol-AS-D-chloroacetate-esterase (CIAE). In the first experiment, Holstein-Friesian cows (n = 204) were used to take two cytology samples at the same time using the conventional cytobrush (CB) and the new cytotape (CT). Both devices were assembled within the same catheter allowing sampling at the same time, and approximately at the same location. Cytotape consisted of a 1.5-cm piece of paper tape rolled on the top of an insemination catheter covered with a double guard sheet. Parameters used to evaluate both methods were: PMNs percentage, total cellularity, quality of the smears, and red blood cell contamination. The concordance correlation coefficient analysis was used to assess agreement between continuous and Pearson chi-square tests for categorical variables. Agreement between the percentage of PMNs in both methods was good ρ = 0.84 (0.79, 0.87) with a minor standard error of 2%. Both methods yielded similar total cellularity (P = 0.62). Cytotape yielded better quality smears with more intact cells (P < 0.01) while samples that were taken by CB were more likely to be bloody (P < 0.01). Hence, CT and CB methods yielded smears with a similar PMNs percentage and a total number of cells, but CT provided smears with higher quality and significantly less blood contamination. For the second experiment, 114 duplicate cytology slides were stained using both Diff-Quik and CIAE. Agreement between PMNs percentage in both staining techniques was good ρc = 0.84 (0.78, 0.89) with a standard error of only 2%. Hence, Diff-Quik was confirmed as an easy, fast, and high-quality staining technique, which can be routinely used to stain endometrial cytology samples satisfactorily.