Equine Ovarian Tissue Research Articles

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy’s (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy’s solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Effect of fixative solutions in equine ovarian tissue

Histological processing is a classical methodology used for an efficient assessment of ovarian tissue quality. Although it is still controversial which is the most appropriate fixative solution for equine ovarian tissue, the choice of an appropriate fixative is critical to evaluate the improvement of assisted reproductive techniques for fertility preservation. This study aimed to evaluate immediate histological processing, based on an ethanol 70% fixative solution for a short-time exposure (ST-EtOH, 45 min), compared with two classically used fixative solutions [Carnoy (CAR) and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h) on equine ovarian tissue. The end points evaluated were morphology (normal and abnormal) and classes (primordial and developing) of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters. Ovaries (n = 6) from ovariectomized young mares (2–8 years) were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1,444 histological slides. High heterogeneity in the number of follicles per individual mare was observed. Equine ovarian fragments fixed in both CAR treatments had a greater (P = 0.014) percentage of morphologically normal primordial and total (primordial and developing classes combined) follicles compared toST-EtOH and both PFA treatments. Although the follicular diameter, regardless of follicular class, was smaller (P = 0.001) in both CAR treatments compared to the other treatments, the nuclear diameter and the nucleus-follicle ratio were greater (P = 0.001) in both CAR treatments compared to the other treatments. A lower (P < 0.001) percentage of morphologically normal developing follicles was observed in ST-EtOH compared to all other treatments. Both CAR treatments had greater (P < 0.001) stromal cell densities than the ST-EtOH and PFA 6h fixatives. In conclusion, Carnoy's solution is the recommended fixative, rather than ST-EtOH and PFA fixatives, for studies requiring more reliable outcomes concerning cellular morphology of equine ovarian tissue. The ST-EtOH fixative proved to be an acceptable alternative to fix equine ovarian tissue when a quick histological evaluation is required for small samples prior to more complicated, time-consuming, and expensive techniques. PFA can be a reasonable fixative for morphological evaluation of equine ovarian tissue, offering the advantage of being suitable for immunohistochemistry techniques since Carnoy's solution is not recommended for this purpose. More studies are required to further understand the effects of different fixative solutions on the morphological ultrastructure and morphometry parameters in different cellular populations of equine ovarian tissue.

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

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 summaryDue 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.

Heterotopic autotransplantation of equine ovarian tissue using intramuscular versus subvulvar grafting sites: Preliminary results

Ovarian tissue transplantation (OTT) is a technique well established and successfully applied in humans using mainly orthotopic or heterotopic transplantation sites. In livestock, OTT is still in its infancy and, therefore, different aspects of the technique, including the efficiency of different heterotopic OTT sites as well as the potential effect of age (i.e., young vs. old mares) in the ovarian graft quality, need to be investigated. The present study investigated the efficacy of the intramuscular (IM) or the novel subvulvar mucosa (SV) heterotopic autotransplantation sites to maintain the survivability of the grafts for 3 and 7 days post-OTT. Ovarian biopsy fragments were obtained in vivo and distributed to the following treatments: Fresh control group (ovarian fragments immediately fixed), SV-3, IM-3, SV-7, and IM-7. During and after graft harvesting, the macroscopic characteristics of the grafts (i.e., adherence, morphology, and bleeding) were scored, and the percentages of morphologically normal and developing preantral follicles as well as the follicular and stromal cell densities of the grafts were evaluated. The results were that similar (P > 0.05) macroscopic scores were observed between both transplantation sites 7 days post-OTT, with positive correlations (P < 0.01) found among adherence, morphology, and bleeding of the grafts. A lower (P < 0.05) percentage of morphologically normal follicles was found 7 days post-OTT in the SV site (82%) compared with the Fresh control group (99%) and IM site (95%); however, the percentages of developing follicles were similar (P > 0.05) between both transplantation sites 7 days post-OTT (30–43%). Although similar (P > 0.05) follicular densities were found in both transplantation sites in young and old mares at 3 and 7 days post-OTT, large individual variation in the follicular depletion rate was observed regardless of transplantation site. The Fresh control group and SV-7 treatments had higher (P < 0.05) stromal cell densities in young and old mares compared with both IM-7 treatments. When comparing transplant sites between young and old mares, the follicular density in old mares and the stromal cell density in young mares were greater (P < 0.05) in the SV than in the IM site. In conclusion, even though the transplantation sites differentially affected some end points, overall comparable findings of the OTT technique using both heterotopic autotransplantation sites (i.e., IM and SV) for equine ovarian tissue were observed.

Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue

Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P > 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.

Genetic variability in equine GDF9 and BMP15 genes in Arabian and Thoroughbred mares

Abstract In horses, multiple ovulation resulting in implantation of multiple embryos is adverse. However, understanding the mechanisms underlying initiation of multiple ovulation (MO) is advantageous and is related to an increase in efficiency of embryo transfer techniques. It has been postulated that MO may have a genetic background. Two major genes: bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are considered to play a crucial role in folliculogenesis and controlling the ovulation rate. Thus, the aim of the presented study was to identify the variation within equine BMP15 and GDF9 genes to verify their potential role on spontaneous, repetitive multiple ovulations in mares. In addition, variation screening of investigated genes in population of Thoroughbred and Arabian breeds was performed together with establishment of transcript abundance of BMP15 and GDF9 genes in equine ovarian tissue. Sanger sequencing of Arabian and Thoroughbred mares divided according to ovulation rate, revealed occurrence of 3 SNPs in BMP15 and STS in GDF9 genes. The PCR-RLFP and statistical analysis indicated that none of the genotype frequencies were significant in any breeds and none of them were claimed as functional according to ovulation rate. Furthermore, evaluation of transcript abundance by RT -PCR of both genes in ovarian tissues showed that expression of both genes was similar but GDF9 was significantly expressed in growing follicles with 21-30 mm diameter and in ovarian parenchyma, which suggest their potential role in folliculogenesis.

Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

Equine ovarian tissue viability after cryopreservation and in vitro culture

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C.

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

Efeito do tipo de fixador e tempo de fixação na morfologia de folículos pré-antrais equinos

&lt;p&gt;O objetivo deste estudo foi investigar a eficácia dos fixadores teciduais Bouin, Carnoy ou Formol 10% em fragmentos ovarianos equinos. Ovários (n=4) de éguas, sem raça definida, foram obtidos de abatedouro local e transportados em recipiente térmico a 20 ºC. Imediatamente após a coleta, os ovários foram lavados com solução de PBS modificado (Cultilab®, Campinas-SP, Brasil), e divididos em nove fragmentos com aproximadamente 5x5x1 mm, retirados do parênquima de cada ovário. Em seguida, os fragmentos ovarianos foram imersos em um dos três diferentes fixadores, Bouin (B), Carnoy (C) ou Formol 10% (F), por 6, 12 ou 24 horas. Cada fragmento foi acondicionado individualmente em um frasco contendo aproximadamente 20 vezes o volume da solução fixadora. Após este período, foram mantidos em álcool 70% por 24 horas. Para cada fixador e tempo foram realizadas quatro réplicas. No processamento histológico, os fragmentos foram desidratados em concentrações crescentes de álcool, diafanizados em xilol e incluídos em parafina. Em seguida, foram feitos cortes seriados de 5 ?m em micrótomo rotativo (Leica®, Wetzlar-Alemanha), seguidos da montagem de lâminas e coloração com ácido periódico de Schiff (PAS) e hematoxilina. Foram avaliadas 540 lâminas com 1.620 cortes histológicos, contendo 465 folículos pré-antrais que foram classificados como íntegros ou degenerados. A degeneração foi detectada pela presença de pelo menos um dos seguintes aspectos: retração do citoplasma, núcleo picnótico, vacúolos citoplasmáticos, deslocamento das células da granulosa e/ou rompimento da membrana basal. Um teste de regressão logística foi utilizado para a análise estatística, e as diferenças foram consideradas significativas quando P&amp;lt;0,05. O fixador Carnoy utilizado por 24 horas proporcionou as melhores condições de integridade morfológica (53,3%; 32/60) em relação aos demais, sendo Boiun por 24 h o tratamento menos eficaz (19,1%; 9/47). Os demais tratamentos apresentaram os resultados a seguir: C12h 50% (30/60), C6h 40% (24/60), F24h 37,8% (17/45), F12h 35,1% (13/37), F6h 32% (16/50), B12h 30,5% (18/59) e B6h 24,4% (11/45). Portanto, sugerimos que a fixação de tecido ovariano equino com Carnoy por 24 horas é o mais indicado para preservação morfológica de folículos pré-antrais.&lt;/p&gt;

44 EFFECT OF CRYOPROTECTANT AGENTS IN EQUINE OVARIAN BIOPSY FRAGMENTS

Studies on ovarian tissue preservation in horses are scarce, and only one research group (Devireddy et al. 2006 Mol. Reprod. Develop. 73, 330–341) has tested the effect of the cryoprotectant agents in equine ovarian tissue. The purpose of this study was to evaluate (1) the effect of cryoprotectant agents in the morphology of equine preantral follicles (PAF) and structure of the ovarian tissue, (2) the relationship between ovarian stromal cell density and PAF density, and (3) the association of ovarian stromal cell density with PAF morphology after exposure to cryoprotectant agents. Three independent experiments with identical methodologies were performed in this study. Each experiment was composed by one cryoprotectant agent (dimethyl sulfoxide, ethylene glycol, propylene glycol) performed in 3 replicates. Ovarian biopsy fragments (1.5 × 1.5 × 10 mm) were harvested from 6 mares and submitted to 4 times of exposure (0, 10, 15, 20 min) at room temperature in a solution of α-minimal essential medium (MEM)+ and one cryoprotectant (dimethyl sulfoxide, ethylene glycol, or propylene glycol) at a concentration of 1.5 M. The cryoprotectant removal was performed by a 3-step (5 min each) equilibration process using (1) α-MEM + 0.5 M sucrose, (2) α-MEM + 0.25 M sucrose, and (3) α-MEM alone after each time of exposure. All fragments were fixed in Bouin’s solution for histological analysis. Data were analysed by ANOVA, Fisher’s exact test, Spearman’s rank correlation, and power test. The PAF density, stromal cell density, and area of the fragments were not affected (P &gt; 0.05) by any of the cryoprotectant agents throughout the time of exposure. However, the morphology of the PAF was affected (P &lt; 0.05) by the cryoprotectant agents. In the propylene glycol and dimethyl sulfoxide, higher (P &lt; 0.05) percentages of abnormal PAF were observed at 10 and 20 min of exposure, respectively. However, the PAF morphology in the ethylene glycol treatments was not affected (P &gt; 0.05) throughout the times of exposure. Strong correlations (range, 0.57 to 0.77; power, 96 to 99%) were identified between PAF density and stromal cell density in all experiments. When stromal cells were classified into groups of density (i.e. low, 25.67–34.66; medium, 34.67–43.66; and high, 43.67–52.67 cells/2500 μm2), only the high stromal cell density was positively correlated (P &lt; 0.001) with the PAF density; however, no correlation between stromal cell density and PAF morphology was observed. In conclusion, (1) ethylene glycol seems to be a less harmful cryoprotectant agent to equine PAF, (2) exposure to cryoprotectant agents did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAF.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM+ supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

99 GLUCOCORTICOID RECEPTORS ARE EXPRESSED IN OVARIES OF NEWBORN AND ADULT FEMALE HORSES

In contrast to other domestic animal species, in vitro maturation (IVM) of oocytes in the horse is still not successful. Oocytes for IVM are obtained either from slaughterhouse ovaries or via ovum pick-up from living mares. Both situations may be associated with a stress-induced glucocorticoid release. So far, neither an involvement of glucocorticoids in follicle and oocyte maturation nor the presence of glucocorticoid receptors (GCR) in ovarian tissue has been investigated in the horse. We hypothesised that GCR are expressed in equine ovarian tissue independent of the animal's age and stage of the oestrous cycle. Ovaries (n = 40) were collected from killed newborn female foals (n = 10) and killed or slaughtered adult mares (n = 10). For assessment of GCR mRNA expression, ovarian samples were fixed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zoeterwoude, the Netherlands) and stored at –80°C. Various cell populations were isolated using laser capture microdissection on cryosections. After RNA extraction, samples were analysed by qualitative RT-PCR and real time-PCR. For analysis of GCR protein, tissue was fixed in Bouin's solution and histological slides immunostained using a monoclonal antibody for GCR (Ab2768, Abcam, Cambridge, UK), followed by visualisation with diaminobenzidine. One tertiary follicle per slide (40×; light microscopy) was analysed and percentages of cells staining positive for GCR calculated. Statistical analysis was done with the SPSS Statistics 21 software (SPSS Inc., Chicago, IL, USA). Expression of mRNA for GCR was detected in oocytes, cumulus cells, granulosa, and theca cells, independent of age and stage of the oestrous cycle. In both neonates and adults, nuclei of the oocytes and cumulus cells stained positive for GCR regardless of stage of folliculogenesis. Also, GCR were constantly expressed in granulosa cells from both preantral and antral follicles. Percentage of granulosa cells staining positive for GCR (adult: 73.6 ± 3.2, fillies: 72.4 ± 1.9%) was higher (P &lt; 0.001) than of theca cells (adult: 56.8 ± 3.9, fillies: 57.2 ± 1.9%), but not affected by age. GCR were lacking in ovarian stroma of adults but not of neonates. In periovulatory follicles from adult mares, GCR were abundant in developing luteal cells. GCR were also detected in the nuclei of luteal cells in corpora haemorrhagica and corpora lutea. Follicular atresia was associated with a decrease of GCR independent of cell type and age. This study describes for the first time the expression of GCR in horse ovaries, which are present independent of age of the animal, stage of folliculogenesis, and oestrous cycle stage. Results suggest that glucocorticoids are involved in follicular and oocyte maturation, ovulation, and luteal function in the horse. Presence of GCR in the ovaries of newborn horses suggests a role of glucocorticoids in ovarian tissue maturation. Nevertheless, detrimental effects of excess glucocorticoid secretion due to stress on follicular development, oocyte maturation, and luteal function cannot be excluded in the mare.

113. Freezing characteristics of macaque and equine ovarian tissue sections in mixtures of dimethyl sulfoxide and ethylene glycol

113. Freezing characteristics of macaque and equine ovarian tissue sections in mixtures of dimethyl sulfoxide and ethylene glycol

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.