Serum Testosterone Levels and Seminal Plasma Proteins in Castrated Stallions P.N. Guasti * , P.M. Papa, R.A. Schmith, L.S. Camargo, L.R.P. Andrade Jr., L.F.M.C. Silva, C.P. Freitas-DellAqua, F.F. Souza, F.O. Papa Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, Unesp, Botucatu, Brazil 1. Introduction Testosterone is the major circulating androgen in males; essen- tial for the development and maintenance of secondary sexual characteristics as well as initiation and maintenance of spermato- genesis [1]. Seminal plasma is a complex fluid composed of secre- tions produced by testes, epididymides and accessory sex glands (AG) and contains several components that play important roles in sperm maturation and fertilization [2]. It is know that the AG epithelium is androgen-dependent; the decrease of the testos- terone levels induces a deficient function of these glands [3] and in rams, causes changes in seminal plasma proteome [4]. The aim of this study was to investigate the serum testosterone levels and expression of seminal plasma proteins after castration in stallions. 2. Materials and Methods Eleven stallions aged 3 to 4 years old were submitted to semen collection with artificial vagina and then to bilateral orchiectomy. After 15 and 30 days of the surgery procedure, animals were again submitted to “semen” collection. For serum testosterone analysis, blood samples were collected by standardized venipuncture technique in all stallions before castration. After castration, blood samples were collected only in animals that were able to finish the ejaculatory process (tail “flagging”, urethral pulsations, treading on hindlimbs, post-ejaculatory relaxation). Serum levels of testosterone were measured by radio immune assay (RIA). For proteomic analysis, semen or seminal fluid samples were centri- fuged at 1 500 Â g for 15 min after collection to remove sperm cells, then the supernatants were centrifuged at 10 000 Â g for 60 min to remove debris. The determination of total protein was performed by BCA-assay. In-solution protein digestion was per- formed using 20 ng/mL trypsin (Promega, sequencing grade, Madison, USA) at a protease: protein ratio of 1:50 (w/w), the resulting peptides were loaded onto 1-mL C18 columns (Sep-Pak, Massachusetts, USA) for clean-up and then analyzed by LC-ESI- QToF-MS/MS. The spectra were acquired using software MassLynx v.4.1 and raw data files were converted to a peak list format (mgf) without summing the scans by the software Mascot Distiller v.2.3.2.0, 2009 (Matrix Science Ldt, London, UK) and searched against the mammalian section of Uniprot_Sprot database. Gene ontology annotationwere analyzed by Strap software (v.1.5; soft- ware for researching annotations of proteins). Protein expression was measured by the EmPai (Exponentially Modified Protein Abundance Index) fold change between groups. Values are pre- sented as mean Æ SEM. The distribution of variables were checked for normality using Kolmorov-Smirnov test. Data normally distributed were tested by unpaired t-test, and those not normally distributed were tested by Mann-Whitney rank sum test. Signifi- cant difference was defined as P < 0.05. 3. Results and Discussion Seven animals ejaculated 15 days after castration; and only one animal ejaculated in D30. The castration reduced serum testos- terone levels from 544.48 Æ 230.07 down to 15.66 Æ 2.48 pg/mL (D15) (P < 0.05). The testosterone concentration in D30 was 11.23 pg/mL. Different proteins were identified among groups, including 15 proteins in seminal plasma, 13 proteins in D15 and 15 proteins in D30 samples. The castration decreased the expression of proteins most commonly related to the reproductive process, such as HSP1, CRISP3 and seminal plasma protein A3. Unexpectedly, kallikrein- 1E2 (KLK1E2) showed increased expression of up to 1.8 fold in D30 compared to seminal plasma. Four proteins (KLK1E2, serum albu- min, clusterin, major allergen Equ-c1) were common to all groups and were involved in binding and catalytic activity. Catalase increased expression 9-fold in D30 compared to D15. Nine proteins were identified only in D30, which were involved in immune sys- tem and metabolic process, angiogenesis and biological adhesion. 4. Conclusion In general, AG conforms to the general pattern of steroid- regulated systems as they are under androgenic control [3], in which hormone withdrawal lead to differential decreases in pro- tein expression. The protein expression in seminal plasma is not immediately terminated after castration, due to residual levels of testosterone. However, the expression of proteins, especially pro- teins related to the reproductive process, seems to be more sen- sitive to the decrease of testosterone and can be possibly used as biomarkers in cases of loss of endocrine function of the testicular parenchyma. Increased expression of KLK1E2 and catalase in cas- trated group is possibly related to decreased overall protein expression rather than actual increase in expression, since some abundant proteins can mask other protein components present at low concentrations [5]. Interestingly, the KLK1E2 and catalase are synthesized by the prostate [6,7], suggesting that the stallion’s prostatic epithelium is more resistant to decreasing testosterone. Contents lists available at ScienceDirect Journal of Equine Veterinary Science journal homepage: www.j-evs.com Journal of Equine Veterinary Science 66 (2018) 64