Immunization of Prepubertal Bitches Against Porcine Zona Pellucida: Effects on Estradiol and Progesterone Profiles
*Pedro L. Lorenzo González, Pilar Millán Pastor, Gema Silván Granado, Irwin K.M. Liu, Juan C. Illera Del Portal
*Dpto. Fisiología Animal - Universidad Complutense De Madrid, Ciudad Universitaria S/N
Madrid, 28040 Madrid ES
Overpopulation of companion animals is a complex societal, ethical, economic and humane problem in both developed as well as developing nations. It is estimated that in Spain each year are abandoned thousands of dogs, and millions are euthanized annually in the U.S. Methods to control overpopulation of companion animals have relied on spay-neuter programs. Alternative programs have focused on immunocontraception and subsequent return to fertility. However, effective immunosterilants will result in a permanent loss of fertility as well as sexual behaviour without adverse side effects related to the animal's health and well being. Among others, immunization methods with GnRH and/or purified zona pellucida may be an alternative in order to reach the immunosterilization. The aim of this study was to elucidate the effects of immunization with porcine zona pellucida (pZP) on gonadal function in the bitch by measuring a) progesterone and estradiol levels, b) antibody titers and, c) sexual behaviour in the animals.
All the experimental procedures were authorized by the Ethics and Animal Experimentation Comitee (Veterinary School, UCM). We used 12 prepubertal Beagle bitches divided in controls (n=4) and problems (pZP group, n=8). After the aclimatation period, they received the following injections: pZP group 25 mg of purified pZP (from Dr. Liu's lab, Dep. Population Health and Reproduction, UCDavis, USA), emulsified with Freund's incomplete adjuvant (1 mL, i.m.). Two boosters were administered after four, and eight weeks from first immunization. Controls received only 1 mL of Freund's adjuvant. Blood samples were taken weekly from radial vein to determine steroid hormones and antibody titers. In addition, vaginal cytology was performed to ascertain the physiological changes in their estrous cycle. Steroid levels (progesterone and estradiol) were analyzed by competitive EIA Silván et al. (Steroids, 34:152, 1993), while antibody titers were determined by direct EIA, as reported by Illera et al. (J. Physiol Biochem, 53:130, 1997). Calculations were made by using Biomedical Data Program.
Hormone levels shown that no alteration was found in the endocrine status of the females, either immunized or not. The results for estradiol showed a physiological preovulatory peak, in the estrous phase. Progesterone levels were also similar in both, pZP and control groups. However, antibody titers showed marked differences when comparing both groups. The dogs received pZP showed high titer of antibodies. In addition, the antibody titer increased when the boosters were injected. Finally, vaginal cytology did not show any differences when comparing two groups.
The results show that the immunization with pZP induce a solid immune response, with no alterations in the endocrine function and estrous cycle in these animals. We postulated that there is a blockade prior to the first appearance of zona pellucida (4 months) during the development of growing follicles. Experiments about fertilization and pregnancy in immunized pZP-group are currently under way.