<div style="text-align: justify;">This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes (325&ndash;500 mm) and treated them with ovine follicle stimulating hormone (OFSH) at different concentrations (0&ndash;400 mIU). Follicle diameter, antrum formation and cumulus oocyte complex (COC) recovery rate were significantly higher (p &lt; 0.05) under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations (100, 200 and 400 mIU). Additionally, follicles cultured for 3 and 4 d differed significantly (p &lt; 0.05) in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250&ndash;300, 301&ndash;400 and 401&ndash;500 mm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250&ndash;300, 301&ndash;400 and 401&ndash;500 mm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250&ndash;300 mm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.&nbsp;</div>

<div style="text-align: justify;">Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes</div>

<div>Background: Ovarian follicular development and growth are controlled by many hormones and growth factors. Despite the fact that LH and estradiol-17&beta; have been utilized for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17&beta; is not known. Therefore this study aimed to investigate the influence of addition of LH and estradiol-17&beta; at different time points on in vitro development of preantral follicles (PFs&rsquo;) in sheep.&nbsp; 
 
Method: Preantral follicles isolated from the ovarian cortical slices using micro dissection method were cultured for six days in Bicarbonate buffered Tissue culture medium 199B (TCM 199B) or in a standard culture medium supplemented with LH (2 &mu;g/ml) and estradiol-17&beta; (5 ng/ml) at different points during the culture period. COCs isolated from the follicles at the end of six day culture in different treatments were subjected to in vitro maturation for additional 24h.&nbsp; 
 
Result: Supplementation of LH and estradiol-17&beta; during last two days of the culture supported better proportion of PFs&rsquo; exhibiting growth whereas supplementation of LH and estradiol-17&beta; during first two days of the culture supported better average increase in diameter and proportion of PFs&rsquo; exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17&beta; to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs&rsquo; in vitro. Following supplementation with LH and estradiol-17&beta; for the first two days culture of PFs&rsquo; in standard medium appears to be advantageous for the development of preantral follicles in vitro.</div>

<div>Effect of LH and Estradiol-17&beta; Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep</div>

Background: The study aimed at quantifying the estradiol 17 &beta; receptor gene in in vivo developed and cultured ovarian follicles of sheep in different culture media by RT-q PCR. Since, estradiol 17 &beta; is a key steroid hormone for antrum formation, preovulatory follicle maturation, expression of genes involved in ovarian differentiation and follicular rupture during ovulation of mammals and the local action of any hormone on the target cell or tissue depends on the expression of its receptor. 
 
Methods: Estradiol 17&beta; receptor mRNA expression in sheep oocytes and cumulus cells was studied using quantitative RT-PCR for: (i) In vivo grown preantral, early antral, antral, large antral follicles and cumulus-oocyte complexes (COCs) obtained from large antral follicles subjected to 24 h of in vitro maturation and (ii) Accordingly, the cumulus cells and oocytes were collected from different development stages of preantral follicles cultured in vitro in TCM199B (Group I), TCM199B +E2 (5 ng/mL) (Group II) and Standard medium + E2 (5 ng/mL) (Group III) for 3 min, two, four or six days and subsequently matured in vitro for 24 h.&nbsp; 
 
Result: Estradiol 17 &beta; receptor was observed at all stages of ovarian follicles in both cumulus cells and oocytes. The upregulation of the ER &beta; gene was noted as the follicle grew in both the media supplemented with estradiol 17 &beta;. Irrespective of in vitro groups, cumulus cells from COCs of the large antral follicles matured in vitro for 24 h exhibited the highest gene expression of the estradiol 17 &beta; receptor. Among the three groups under study, the follicular cells of PFs&rsquo; cultured in group III showed the highest expression of the ER&beta; gene at any point of their development. The supplementation of estradiol 17 &beta; in low doses stimulated synergism with growth factors and hormones to express its receptors in cultured preantral follicles of sheep.

Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes

Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

Quantification of Estradiol 17β (Erβ) Receptor Gene in Cultured and in vivo Grown Ovarian Follicles of Sheep