Document Type : Research Paper


Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq


The current study aims to investigate the role of steroid-free follicular fluid antiserum on ovaries morphometric growth and development in virgin female rats at two sequential estrous cycles. Bovine follicular fluid aspired from graafian follicles, steroids were removed by treatment with activated charcoal, in order to obtained steroid-free bovine follicular fluid (S-FBFF) which was used for immunization of male rabbits to prepare S-FBFF antiserum (S-FBFF-ab). Sixty virgin female rats were assigned into control and treatment groups (30 each), intraperitoneal injected with a single dose of distilled water 100µl/rat and S-FBFF-ab 100µl/rat at late metestrus, respectively. At the estrus phase of the first and second estrous cycle, 15 females from each group of each cycle were anesthetized and ovarian samples were obtained for histological examination. In comparison with control, the results of S-FBFF-ab treated female rats revealed a significant increase of relative ovaries and uteri weights at both estrous cycles. Morphometric examination showed progressive ovarian proliferation at the first estrus phase in S-FBFF-ab treated female rats through elevation of the number of primaries, graafian, and total follicles. In conclusion, passive immunization against endogenous inhibin using S-FBFF-ab could augment the reproductive fecundity through increase ovarian growth and development.


The present findings revealed an increase of female reproductive fecundity through augmentation of female reproductive endocrine activity.


Main Subjects

  1. Ishigma H, Medan MS, Watanabe G, Shi Z, Kishi H, Arai KY. A new alternative method for superovulation using passive immunization against inhibin in adult rats. Biol Reprod. 2004;71:236-243. 10.1095/biolreprod.104.027789
  2. Yan L, Li H, Shi Z. Immunization against inhibin improves in vivo and in vitro embryo production laboratory of animal breed improvement and reproduction, institute of animal science, Jiangsu academy of agricultural sciences, Nanjing, China. Anim Reprod Sci. 2015;163:1-9. 10.1016/j.anireprosci.2015.11.001
  3. Al-Sa’aidi JA, Majhwol EM. Effect of immunization against steroid-free bovine follicular fluid on reproductive hormones profile in cycling female rats. Asian J Anim Sci. 2017;11:183-188. 10.3923/ajas.2017.183.188
  4. Al-Sa’aidi JA, Majhwol EM. Pituitary FSHβ and GH and Ovarian GH-r, IGF-1 and IGF-2 gene expression levels in cycling female rats immunized against steroid-free bovine follicular fluid antisera. Asian J Applied Sci. 2017;5(4):770-775.
  5. Kafaji SS, Al-Sa’aidi JA, Khudair KK. Reproductive hormones profile of Iraqi awassi ewes immunized against synthetic inhibin-α subunit or steroid-free bovine follicular fluid. Iraqi J Vet Sci. 2017,31(2):123-128. 10.33899/ijvs.2017.145609
  6. Al-Sa’aidi JA, Khudair KK, Khafaji SS. Reproductive fecundity of Iraqi awassi ewes immunized against synthetic inhibin-α subunit or steroid-free bovine follicular fluid. Asian-Australasian J Anim Sci. 2018;31(8):1169-1175. 10.5713/ajas.17.0660
  7. Ishigame H, Medan MS, Wang H, Watanabe G, Kishi H, Arai KY. Induction of superovulation by immunoneutralization of endogenous inhibin in immature rats. J Reprod Dev. 2005;51:559-66. 10.1262/jrd.17020
  8. Medan MS, Nambo Y, Nagamine N, Shinbo H, Watanabe G, Groome N, Taya K. Plasma concentrations of ir-inhibin, inhibin A, inhibin pro-alphaC, FSH, and estradiol-17beta during estrous cycle in mares and their relationship with follicular growth. Endocrin Rev. 2004;25(1):7-14. 10.1385/endo:25:1:07
  9. Gordon A, Aguilar R, Garrido JC, Guil LS, Sanchez CR, Millan Y, Watanabe G, Taya K, Martin MJ, Sanchez JE. Immunoneutralization of inhibin in cycling rats increases follicle-stimulating hormone secretion, stimulates the ovary and attenuates progesterone receptor-dependent preovulatory luteinizing hormone secretion. Neuroendocrinol. 2010;91:291-301. 10.1159/000299791
  10. Du AG, Wu ZH, Zhang CY, Zhang HY, Liu HM, Chen ZY, Zeng XY. The effect of immunization against Xinjiang fine wool sheep inhibin on the reproductive traits of rats. Afr J Agric Res. 2011;6:5040-5046. 10.5897/ajar10.1084
  11. Takeo T, Nakagata N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLOS ONE. 2015;10(5):1-11. 10.1371/journal.pone.0128330
  12. Findlay JK. An update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biol Reprod. 1993;48:15-23. 10.1095/biolreprod48.1.15
  13. Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. J Biochem. 2010;147:35-51. 10.1093/jb/mvp148
  14. Kumanov P, Kalyana C, Nandipati K, Tomova A, Robeva R, Agarwal A. Significance of inhibin in reproductive pathophysiology and current clinical applications. Reprod BioMed. 2005;10(6):786-796. 10.1016/S1472-6483(10)61124-8
  15. Mather JP, Moore A, Li, R. Activins, inhibins, and follistatins: further thoughts on a growing family of regulators. Proc Exp Biol Med. 1997;215:209-222. 10.3181/00379727-215-44130
  16. Andreone L, Velasquez EV, Abramovich D, Ambao V, Loreti N, Croxatto HB, Parborell F, Tesone M, Campo S. Regulation of inhibin/activin expression in rat early antral follicles. Mol Cell Endocrinol. 2009;309:48-54. 10.1016/j.mce.2009.05.003
  17. Woodruff AK, Krummen LA, Lyon RJ, Stocks DL, Mather JR. Recombinant human inhibin A and recombinant human activin A regulate pituitary and ovarian function in the adult female rat. Endocrinol. 1993;132:2332-73. 10.1210/endo.132.6.8504739
  18. Wise T. Biochemical analysis of bovine follicular fluid: Albumin, total protein, lysosomal enzymes, ions, steroids and ascorbic acid content in relation to follicular size, rank, atresia classification and day of estrous cycle. J Anim Sci. 1987;64: 1153-1169. 10.2527/jas1987.6441153x
  19. Luna LG. Manual of histological staining methods. 3rd ed. Sydney: McGraw Hill Book Company; 1968. 258 p.
  20. Schefler WC. Statistics for biological sciences. 2nd ed., Addison-Wesley Publ. co., Reading MA, USA. 230 p.
  21. Hillier SG, Yong E, Illingworth PJ, Baird DT, Schwall RH, Mason AJ. Effect of recombinant activin on androgen synthesis in cultured human thecal cells. J Clin Endocrinol Metab. 1991;72:1206-1211. 10.1210/jcem-72-6-1206
  22. Rozell TG, Okrainetz RJ. FSH: one hormone with multipule forms, or a family of multiple hormones. In: Chedrese ED (editor). Reproductive endocrinology, A molecular approach. Saskatoon: Springer Science and Business Media; 2009. 144-160 p. 10.1007/978-0-387-88186-7_14
  23. Bhardwaj A, Nayan V, Parvati M, Gupta AK. Inhibin: A role for fecundity augmentation in farm animals. Asian J Anim Vet Adv. 2012;7:771-189. 10.3923/ajava.2012.771.789
  24. Al-Sa’aidi JA, Al-Jayashi GS. Adenohypophyseal immunohistochemical expression levels of FSHβ in cyclic virgin female rats treated with steroid free bovine follicular fluid antiserum. Online Int Interdicip Res J. 2016;6:18-31. 10.29079/vol17iss2art502
  25. Al-Sa’aidi JA, Al-Jayashi GS, Khafaji SS. Ovarian immunohistochemical expression of estradiol 17β in cyclic female rats treated with steroid free bovine follicular fluid antiserum. Al-Qadisiyah J Vet Med Sci. 2018;17(2):35-42. 10.29079/vol17iss2art502
  26. Murthy RM. Effect of Allium cepa extracts on the estrous cycle and ovarian activity in albino mice. Plant Med. 1997;20(5):117-120. 10.1590/s0036-46652011000300007
  27. Bary GA, Saiduddin S, York DA, Swerdloff RS. Effect of estradiol on uterine weight, thyroid function food intake, and pituitary weight of genetically obese (fatty-Zucker) and lean rats. Proc Soc Exp Biol Med. 1976;153(1):88-91. 10.3181/00379727-153-39486
  28. Medlock KL, Forrester TM, Sheehan DM. Progesterone and estradiol interaction in the regulation of rat uterine weight and estrogen receptor concentration. Proc Soc Exp Biol Med. 1994;205(2):146-153. 10.3181/00379727-205-43690
  29. Al-Sa’aidi JA, AL-Okaily BN, Al-Shwilly HA. Uterine implantations and litter size alternation in anti- inhibin and eCG-hCG treated virgin pregnant female rats. Online Int Interdicip Res J. 2016;6(1):36-46. 10.29350/jops.2018.23.2.732
  30. Bley MA, Simon JC, Estevez AG, de Asua LJ, Baranao JL. Effect of follicle stimulating hormone on insulin-like growth factor- I-stimulated rat granulosa cell deoxyribonucleic acid synthesis. Endocrinol. 1992;131:1223-1229. 10.1210/en.131.3.1223
  31. Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR. Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells. Biol Reprod. 1997;56:891-897. 10.1095/biolreprod56.4.891
  32. Findlay JK, Drummond AE, Dyson M, Baillie A, Ethier JF. Production and actions of inhibin and activin during folliculogenesis in the rat. J Mol Cell Endocrin. 2001;180:139-144. 10.1016/s0303-7207(01)00521-4
  33. Al-Sa’aidi JA, Baqir SM. Effect of passive immunization against inhibin-a subunit and non-steroid bovine follicular fluid (NSBFF) on mammary gland growth and development in primiparous female Wistar rats. Reprod Dom Anim. 2012;47(4):416-613. 10.1111/j.1439-0531.2012.02119.x 
  34. Kenny HA, Woodruff TK. Follicle size class contributed to distinct secretion patterns of inhibin isoforms during the rat estrus cycle. J Endocrinol. 2005;10:1210-1242. 10.1210/en.2005-0242
  35. Ko Y, Choi I, Green ML, Simmen FA, Simmen RC. Transient expression of the cytochrome P450 aromatase gene in elongating porcine blastocysts is correlated with uterine insulin-like growth factor levels during peri-implantation development. Mol Reprod Develop. 1994;37:1-11. 10.1002/mrd.1080370102
  36. Kipp JL, Kilen SM, Bristol GS, Woodruff TK, Mayo KE. Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary. J Endocrinol. 2007;148(5):1968-1976. 10.1210/en.2006-1083
  37. Medan MS, Watanabe G, Taya K. Inhibin: Regulation of reproductive function and. practical use in females. Anim Sci J. 2007;78:16-27. 10.1111/j.1740-0929.2006.00399.x
  38. Al-Sa’aidi JA, Samir MS. Effect of passive immunization against inhibin alpha subunit on ovarian growth and development in immature female Wister rats. 14th Scientific Congress of Faculty of Medicine; 2010, Assiut, Egypt.
  39. Al-Sa’aidi JA, Al-Okaily BN, Al-shwilly HA. Comparative effect of anti-inhibin and eCG-hCG supplementation on reproductive hormonal profile in virgin cycling female rats. Al-Qadisiyah J Pure Sci. 2018;23(2):1-8. 10.29350/jops.2018.23.2.732
  40. Al-Sa’aidi JA, Al-Asadi SH. Quantification of mammary Gh-r gene and GH expression in pregnant, delivered and lactating Wister female rats passively immunized at pregnancy against inhibin-α, βa, or βb subunits. Int J Biol Pharm Allied Sci. 2016;5(9):2070-2083.
  41. Al-Sa'aidi JA, ad Thanoon HB. Hypothalamic GHRH and pituitary GH gene expression levels in neonatal inibin immunoneutralized female rats. Aspire. The 5th Congress of the Asia Pacific Initiative on Reproduction; 2014, Brisbane, Australia.
  42. Al-Sa’aidi JA, Al-Kalby JM, Al-Saeedi MJ. Effect of passive immunization against inhibin-A, -BA and -BB subunits on serum growth and differentiation hormones profile in pregnant and lactating primiparous Wister rats. Basrah J Vet Res. 2015;14(1):79-96. 10.33762/bvetr.2015.99877
  43. Demeestere I, Gervy C, Centner J, Devreker F, Delbaera A. Effect of insulin like growth factor-1 during preantral follicular culture on steroidogenesis, in vitroin oocyte maturation and embryo development in mice. Biol Reprod. 2004;70:1664-1669. 10.1095/biolreprod.103.023317