The extracts were pooled and dried completely by a vacuum centrifuge

The extracts were pooled and dried completely by a vacuum centrifuge. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development. Keywords: Yak, Follicular Fluid, Two-dimensional Gel Electrophoresis, Mass Spectrometry, Western Blot == INTRODUCTION == Yaks (Bos grunniens) are mainly distributed in the Qinghai-Tibetan Plateau and nearby regions; they provide meat, milk, wool and fuel for Tibetan and nomadic pastoralists. These animals are essential for agricultural production in high altitude environment regions. Their natural habitat ranges from between 2, 500 to 6, 000 m above sea level. Yaks are seasonally polyestrous, exhibiting cyclic ovarian activity and estrus from July to November (Sarkar and Prakash, 2005; RAF709 Sarkar et al., RAF709 2008). The length of the estrous cycle lasts from 19 to 21 days. However , yaks live in an extremely harsh environment characterized by low temperature, low oxygen content, and high altitude; seasonal nutritional deficiencies seriously affect their reproductive efficiency (Liu et RAF709 al., 2012). Unlike bovine females, yak cows rarely express estrus immediately after post-calving during the breeding season. The annual RAF709 pregnancy rate of most yak breeds is 40% to 60%, as only a small proportion of the cows return to estrus in the first breeding season after calving; most come into estrus in the second or third years under traditional management systems (Zi, 2003). These long postpartum anestrous intervals eventually result in poor reproductive efficiency. Follicular fluid (FF) is a liquid in the ovarian follicle which fills the follicular antrum and surrounds the ovum. FF contains several blood plasma protein components derived from constituents that cross the blood-follicular barrier and several granulosa/theca proteins derived from granulosa and thecal cells (Nandedkar et al., 1992; Fortune, 1994). Thus, FF components may also provide information related to ovarian follicle development. Follicle development is closely associated with yak reproduction and FF provides a very important microenvironment for oocyte development. A previous analysis of FF protein composition aimed to identify molecules which may help follicular development and therefore assist in oocyte selection processes (Schweigert et al., 2006). The study of FF protein components have been made by detecting specific proteins and correlating them to estrus and oocyte quality. Recently, more complex molecular techniques have been introduced, allowing for simultaneous study of dozens of proteins and peptides in biological fluid, in a field known as proteomics (Revelli et al., 2009). Protein composition is very complicated and dynamic, and is therefore a major problem in proteomic analysis of complex biological fluids; consequently, sensitive and high-resolution protein separation techniques are needed. The most popular FF proteonomic approach is presently based on two-dimensional gel electrophoresis (2-DE) followed by protein digestion and mass spectrometry (Grg et al., 2004). At the same time, Western blotting has been used to detect specific proteins in an extracted sample. Recently, easier techniques have been proposed and applied towards studying FF proteins based on protein prefractionation by isoelectric focusing (IEF) as well as nanoliquid chromatography and mass spectrometry (Hanrieder et al., 2008). In order to improve the efficiency of yak breeding, comparative FF proteomics was applied to pHZ-1 analyze different follicular diameters during estrus. This technical scheme involved 2-DE in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blotting. == MATERIALS AND METHODS == == Collection of follicular fluid == Yak ovaries were obtained from a designed abattoir after slaughter in September 2014 in Qinghai Province of China. Sixty ovaries in total were collected from 30 non-pregnant yaks. The yaks were between 6 to 7 years of age, healthy, and free from any anatomical reproductive disorders; they resided 3, 200 RAF709 m above sea level. The FF was directly aspirated from the ovaries by a sterile syringe and then divided into two.