Apoptotic Cell Localization in Preantral and Antral Follicles in Relation to Non-cyclic and Cyclic Gilts D Phoophitphong 1,2 , S Srisuwatanasagul 3 , S Koonjaenak 4 and P Tummaruk 1 1 Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand; 2 Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand; 3 Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand; 4 Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand Contents The objective of this study was to determine apoptotic cell localization in preantral and antral follicles of porcine ovaries. Additionally, the proportion of cells undergoing apoptosis was also compared between delayed puberty gilts and normal cyclic gilts. Ovarian tissues were obtained from 34 culled gilts with age and weight of 270.1  3.9 days and 143.8  2.4 kg, respectively. The gilts were classified according to their ovarian appearance as ‘non-cyclic’ (n = 7) and ‘cyclic’ (n = 27) gilts. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay was used to determine apoptotic cell expression in different compartments of the ovarian tissue sections. All apparent preantral (n = 110) and antral (n = 262) follicles were evaluated using image analysis software. It was found that apoptotic cells were expressed in both granulosa (22.2%) and theca cell layers (21.3%) of the follicles in the porcine ovaries. The proportion of apoptotic cells in the granulosa layer in the follicles was positively correlated with that in the theca layer (r = 0.90, p < 0.001). Apoptosis did not differ significantly between preantral and antral follicles in either granulosa (27.8% and 26.4%, p > 0.05) or theca cell layers (28.6% and 26.5%, p > 0.05). The proportion of apoptotic cells in non-cyclic gilts was higher than cyclic gilts in both granulosa (31.7% and 22.6%, p < 0.001) and theca cell layers (34.8% and 20.2%, p < 0.001). This study indicated that apoptosis of the granu- losa and theca cell layers in the follicles was more pronounced in the ovarian tissue of delayed puberty gilts than cyclic gilts. This implied that apoptosis could be used as a biologic marker for follicular development/function and also that apoptosis was significantly associated with anoestrus or delayed puberty in gilts, commonly observed in tropical climates. Introduction Apoptosis, or programmed cell death, occurs during folliculogenesis of ovarian tissue. Matsuda-Minehata et al. (2006) stated that more than 99% of mammalian follicles, including in pigs, undergo degeneration or atresia during follicular growth and development due to granulosa cell apoptosis. The period of follicle develop- ment during the preantral-to-early antral stage is more susceptible to atresia than the later stage of follicles (Orisaka et al. 2009). In general, granulosa cells are closely associated with oocyte development through their endocrine and paracrine signalling (Goto et al. 2013; Yang et al. 2013). Therefore, granulosa cell dysfunction might lead to poor oocyte quality and infertility (Gilchrist et al. 2004; Su et al. 2009). More- over, granulosa cell apoptosis has been reported to be a cause of follicular atresia in mammalian ovaries (Yang et al. 2013), which leads to infertile animals. To date, various techniques have been used to assess apoptotic cells in many types of tissues. Over the past decades, the detection of DNA fragmentation has been widely used to determine the presence of apoptotic cells in histologic sections (Stadelmann and Lassmann 2000). The termi- nal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay is a method frequently applied to detect DNA fragmentation in tissue sections, including in pigs (Durlej et al. 2012). In pigs, the number of ovulations (i.e. litter size at birth) is one of the most important criteria determining reproductive performance (Tummaruk and Kesdangsakonwut 2015). Due to the female puberty gradation and physiologic condition, folliculogenesis and its regulation are impor- tant for female pigs to regulate many reproductive functions including puberty attainment, oestrous beha- viour and ovulation (Brussow et al. 2002; Ratky et al. 2005; Schwarz et al. 2008). Tummaruk et al. (2009a) reported that a high proportion of gilts in the tropical country of Thailand were culled due to anoestrus, and greater than 50% of those culled had non-cyclic ovaries. Of the gilts culled due to reproductive problems, ovarian abnormalities, such as ovarian cysts, ovarian adhesions, unilateral small ovary and ovo-testis, were reported as causes (Tummaruk et al. 2009a). These findings indicate that ovarian dysfunction in gilts is one of the most common reasons leading to culling of replacement gilts in tropical climates. Therefore, additional knowledge of porcine ovarian function and pathology, as well as its associated factors, is needed. Follicular development in cyclic gilts is different from non-cyclic gilts. In cyclic gilts, follicle development occurs under the influence of corpus luteum (CL) development, while this is not the case in non-cyclic gilts (Murphy et al. 2001). Further- more, the number of ovulations in pigs is also associated with the number of large follicles present during follicle development (Driancourt et al. 1992). Nevertheless, follicles selected to develop until ovulation may not always provide healthy oocytes. Therefore, a biologic marker indicating the competency of follicles in porcine ovaries should be carefully determined to be used as an Reprod Dom Anim 51, 400–406 (2016); doi: 10.1111/rda.12693 ISSN 0936–6768 © 2016 Blackwell Verlag GmbH