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Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans. Lay summary Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Ovogenesis and foliculogenesis in the mare differ from other farm animals and therefore, gamete manipulation, in vitro fertilization and embryo transfer have been very difficult. The histologic traits of ovaries from 12 mares, in estrus and diestrus were studied. Number and size of follicules and corpora lutea were recorded. The ovarian stroma was evaluated using the pricosirius technique for collagen. A simple morphometric analysis was done using computerized scanner programs. During late estrus, one dominant follicle (46 ± 4mm) is seen. The surrounding stroma contains collagen I and III. By the end of the estrus, one hemorrhagic follicle is seen, plus one or two small follicle (2 mm in size). In early diestrus there is a corpus luteum (43-60 mm) and some antral follicles (6±1). Collagen I forms strands inside the corpus luteum and predominates in the perifollicular ovarian stroma. Modifications of the extracellular matrix may change cell function by way of integrines, the matrix being in turn modified by hormones and other tissular factors. Estrogens can be related to collagen III predominance in ovarian stroma whereas progesterone is associated with increased collagen I. Therefore, follicular-stromal interactions are important in ovarian histophysiology. La ovogénesis y foliculogénesis en la yegua, difiere de otros animales de granja, y por lo tanto, la manipulación de gametos, fertilización in vitro y transferencia de embriones ha sido muy dificultoso. Se estudiaron las características histológicas de ovarios de 12 yeguas durante el estro y el diestro. Se registróel número y tamaño de los folículos y cuerpos lúteos. Se evaluó el estroma ovárico usando la técnica picrosirius para colágeno. Se realizó un análisis morfométrico simple utilizando un programa escáner computarizado. Durante el estro tardío, se observó un folículo dominante (46 ± 4 mm). El estroma circundante contiene colágeno I y III. Para el final de este período, se aprecia un folículo hemorrágico, más uno o dos pequeños folículos (2 mm de tamaño). En diestro temprano, hay un cuerpo lúteo (43 - 60 mm) y algunos folículos antrales (6 ±1). El colágeno I forma filamentos dentro del cuerpo lúteo y predomina en el estroma ovárico perifolicular. Modificaciones en la matriz extracelular pueden cambiar la función celular vía integrinas, la matriz está a su vez modificada por las hormonas y otros factores tisulares. Los estrógenos se pueden asociar a la predominancia de colágeno III en el estroma ovárico, mientras que la progesterona se asocia con un aumento de colágeno I. Por lo tanto, las interacciones estroma-folículo son importantes en la histofisiología del ovario.

La ovogenesis y foliculogenesis en la yegua, difiere de otros animales de granja, y por lo tanto, la manipulacion de gametos, fertilizacion in vitro y transferencia de embriones ha sido muy dificultoso. Se estudiaron las caracteristicas histologicas de ovarios de 12 yeguas durante el estro y el diestro. Se registroel numero y tamano de los foliculos y cuerpos luteos. Se evaluo el estroma ovarico usando la tecnica picrosirius para colageno. Se realizo un analisis morfometrico simple utilizando un programa escaner computarizado. Durante el estro tardio, se observo un foliculo dominante (46 ± 4 mm). El estroma circundante contiene colageno I y III. Para el final de este periodo, se aprecia un foliculo hemorragico, mas uno o dos pequenos foliculos (2 mm de tamano). En diestro temprano, hay un cuerpo luteo (43 - 60 mm) y algunos foliculos antrales (6 ±1). El colageno I forma filamentos dentro del cuerpo luteo y predomina en el estroma ovarico perifolicular. Modificaciones en la matriz extracelular pueden cambiar la funcion celular via integrinas, la matriz esta a su vez modificada por las hormonas y otros factores tisulares. Los estrogenos se pueden asociar a la predominancia de colageno III en el estroma ovarico, mientras que la progesterona se asocia con un aumento de colageno I. Por lo tanto, las interacciones estroma-foliculo son importantes en la histofisiologia del ovario.