Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10(6) sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.

Expanded equine cumulus-oocyte complexes exhibit higher meiotic competence and lower glucose 1 consumption than compact cumulus-oocyte complexes To test this 24 hypothesis eCOCs, cCOCs, and expanded or compact mural granulosa cells (EC and CC respectively) were 25 matured in vitro for 30 hours and the maturation rate, glucose metabolism, and expression of genes involved in 26 glucose transport, glycolysis, apoptosis and meiotic competence were determined. Significant differences were 27 found between eCOCs and cCOCs maturation rates (50% vs. 21.7 %; n = 192 and 46 respectively, p < 0.001), 28 glucose consumption (1.8 ± 0.5 vs. 27.9 ± 5.9 nmol/COC; mean ± SEM), pyruvate production (0.1 ± 0.0 vs. 2.4 ± 29 0.8 nmol/COC; mean ± SEM) and lactate production (4.7 ± 1.3 vs. 64.1 ± 20.6 nmol/COC; mean ± SEM) 30 respectively (p < 0.05). Moreover, similar glucose consumption was observed for EC and CC. Hyaluronan 31 binding protein (TNFAIP6) expression was increased in eCOCs and EC, solute carrier family 2 (facilitated glucose 32 transporter) member 1 (SLC2A1) was increased in eCOCs, while glycolysis-related enzymes and solute carrier 33 family 2 (facilitated glucose transporter) member 3 (SLC2A3) expression did not vary between COCs or mural 34 granulosa cell type. Our data demonstrate that metabolic and genomic differences exist between eCOCs and 35 cCOCs and mural granulosa cells in the horse. 36 37Additional Keywords: in vitro maturation, glycolysis, nuclear magnetic resonance, horse. 38 39

Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio ). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.

Clinical examination procedures (CEPs) are cornerstone clinical skills for veterinary practitioners, being taught in all veterinary faculties. CEPs include innocuous procedures that are well tolerated by animals as well as more distressful and less tolerated ones. In a classical approach, institutional animals are used to teach and practice CEPs. Two hundred and thirty-one undergraduate students from four consecutive years were assigned to two groups that used institutional animals only (AO) or a combination of students’ owned animals and simulation models (model–animal, MA) to teach and practice CEPs. This latter comprised stuffed teddy dogs, eye and ear models made of molding silicone, as well as skin models. The learning outcome of each system was compared through questionnaires (throughout classes and at the end of course), grades, and pass rates in objectively structured clinical examinations. Most veterinary students had their own animals, being easy to have a dog per group of two students in class. All the students’ owned animals adapted well to this environment. The interest in the practical activities with the simulation models was comparable to that exhibited in the classical AO system. Students reported to learn more with the MA system than with the AO, while the interest on the subjects and the relevance were appraised similarly in both systems. No differences existed in the final grades and pass rates. The MA system was effective for learning CEPs. Beyond animal welfare advantages, this system increased the out-of-school training and had financial saving benefits, being a valuable option for the teaching and training of CEPs.

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