The aim of this study was to evaluate ovarian tissue pre-treatment with 50 µM Trolox followed by heterotopic transplantation in squirrel monkeys (Saimiri collinsi) and to assess tissue functionality via immunohistochemical analysis of the stroma and ovarian follicles. Five healthy and sexually mature squirrel monkey (Saimiri collinsi) females were used. Heterotopic autografting of fresh ovarian tissue with or without previous exposure to the antioxidant Trolox was performed and grafts were recovered for analysis 7 days later. Tissue vascularisation was confirmed by both macroscopic inspection and cluster of differentiation 31 (CD31) staining. Trolox prevented massive follicular activation and kept the percentages of morphologically normal follicles higher than in untreated grafts. Expression of anti-Müllerian hormone in developing follicles was observed only in controls and Trolox-treated grafts. Also, immunostaining for growth differentiation factor-9 was positive only in primordial follicles from controls and from Trolox-treated grafts. Although Trolox improved follicular quality and avoided apoptosis in stromal cells, ovarian tissue fibrosis was increased in Trolox-treated grafts, mainly due to an increase in collagen Type I synthesis.

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS+50 μM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS+10 ng/ml selenium. Ovarian fragments were subsequently frozenthawed in the presence of FS with or without 50 μM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Troloxfree FS compared with FS+50 μM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 μM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.

Endocrine monitoring of non-human primates (NHP) via faecal metabolites of steroid hormones appears as a useful non-invasive alternative to evaluate the reproductive status of free living NHP, as well as of those kept in captivity but of difficult handling. However, validation is needed with plasma values before its application in the field. The aim of the present study was to monitor the different phases of the menstrual cycle from the new world NHP Sapajus apella and S. libidinosus. For this, hormonal and faecal plasma levels of E 2 , P 4 and cortisol were assessed during different days of the menstrual cycle, together with colpocitology. The mean duration of the menstrual cycle according colpocitology was of 21.7 and 21.0 days for S. apella and S. libidinosus, respectively. These values were similar to those observed via plasma analysis, i.e. 22.7 and 20.3 days for S. apella and S. libidinosus, respectively. The day of plasmatic E 2 peak was set as Day -1 and the estimated day of ovulation was set as Day 0 and occurred two days earlier in S. libidinosus than in S. apella females. In both species, it was observed a delay in faecal E 2 peak of six days for S. apella and of 11 days for S. libidinosus when compared with the plasma peak. A maximum P 4 plasma concentration was observed in the middle of luteal phase in S. apella and in S. libidinosus, both at around day 5 . However, faecal P 4 peaks were detected at days 9 and 8 in S. apella and S. libidinosus , respectively. Mean plasma and faecal cortisol levels were variable during all ovulatory cycle of S. apella and S. libidinosus females. Although no exact correlation was observed between plasmatic and faecal profile of steroid hormone, faecal samples were able to indicate ovarian cycle phase, being important to assess the reproductive status of the females applying a non-invasive method.