Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing.

Purpose: To investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways. Methods: The bAM were acquired from normal full-term births (n = 10), processed, and stored at -80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography-mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM. Results: The analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways. Conclusions: The high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.

Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares.

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Alguns aspectos biométricos do aparelho genital externo de jumentos doadores de sêmen da raça Pêga / Some biometric aspects of the external genital tract from donkey Pêga breed semen donors

O presente estudo foi conduzido com seis jumentos reprodutores da raça Pêga, com peso médio de 272±34,9 (231-326kg), sendo subdivididos em adultos (J1, J2 e J3, média 15 anos) e jovens (J4, J5 e J6, média 3,5 anos). Os reprodutores apresentavam histórico normal de fertilidade à monta natural e, após realização de exame andrológico, foram classificados como aptos à reprodução. Foram realizadas 180 coletas de sêmen. Os reprodutores apresentaram parâmetros seminais considerados normais para a espécie. De agosto/2006 a fevereiro/2007, após a realização do exame andrológico, cada reprodutor foi submetido a três ou quatro mensurações do aparelho genital externo para: comprimento testicular (CT - crânio caudal), altura testicular (AT - dorso ventral), largura testicular (LT - médio lateral) e espessura do funículo (FUN - terço médio). Os dados de biometria testicular foram utilizados para o cálculo do índice testicular (IT) e volume testicular (VT). O IT médio observado foi de 7,4 (5,9 a 9,6), e o VT esquerdo foi de 155,5±14,4mL, enquanto o direito foi de 149,3±14,9. A medida de FUND foi de 25,3±3,3mm (20,3a 30,8mm), e de FUNE foi 24,6±1,5mm (22 a 26mm). Jumentos, mesmo com IT menor do que o proposto para garanhões; se apresentaram mais eficientes que os últimos para a produção espermática. O volume espermático, associado ao IT, pode ser usado como ferramenta auxiliar na estimativa da constatação de normalidade para testículos de jumentos. A mensuração in vivo do funículo espermático pode ser uma nova variante na utilização do exame andrológico de jumentos. Os dados descritos podem servir de valores de referência e valores práticos quanto à biometria do aparelho genital externo de jumentos.

The present study was performed with six Pêga breed donkeys, weight from 231 to 326kg (272±34.9). The breeders were divided into the adult group (J1, J2 e J3, 15 years old average) and the young group (J4, J5 e J6, 3.5 years old average). . The donkeys presented normal rates of fertility at natural mating. After breeding soundness evaluation all animals were considered capable for reproduction. A hundred eight semen collections were realized, and the donkeys presented normal seminal parameters for the specie. From August/2006 to February/2007, after semen collection each donkey was submitted to three or four measurements of the extern genital tract (both right and left side) for: testis length (LT - cranio caudal); testis height (TH - dorso ventral) and thickness of spermatic funiculum (FUN - third part). The biometric data from biometric testis were used to calculate the testis indices (TI) and testis volume (TV). The TI observed was 7.4 (5.9 a 9.6) and TV was 155.5±14.4, 149.3±14.9 respectively left and right. The average FUN was 25.3±3.3mm (20.3 a 30.8mm) to the right and 24.6±1.5mm (22 a 26mm) to the left side. The donkeys presented lesser TI than those proposed for the stallions, but the asinine were more efficient for sperm production than the average reported to stallions . The testis volume associated with IT can be used as an additional tool to estimate the normality of the breeder's testis. The measurement in vivo of spermatic funiculum can be used as a new component for the breeding soundness evaluation of donkeys. The genital extern tract biometric values can support routine breeding evaluation and can be applied to news studies.