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Virginia-Maryland Regional College of

Veterinary Medicine

Fertility in male dogs vaccinated with a GnRH vaccine containing a B-cell adjuvant Lara A. Brennecke, Leeah Chew, Clifton Cassidy, Beverly J. Purswell, Stephen M. Boyle

V I R G I N I A P O L Y T E C H N I C I N S T I T U T E A N D S T A T E U N I V E R S I T Y, B L A C K S B U R G, V A The objective of this study was to evaluate the short-term effect on fertility of a B cell adjuvant added to a GnRH vaccine given to male dogs. The aims were to assess quantitative differences in serum GnRH antibody titers, serum testosterone levels and semen qualities over six weeks post vaccination. Male dogs (n=20) were divided into four treatment groups: group one was administered one dose of canine gonadotropin releasing factor immunotherapeutic (cGRFI) (Pfizer) on day 0, followed by a cGRFI booster on day 14; group two was administered one dose of cGRFI with Sigma Adjuvant System on day 0, followed by a saline injection on day 14; group three was administered one dose of cGRFI with flagellin on day 0, followed by a saline injection on day 14; group four was administered a saline injection on day 0 and 14. Blood samples were collected weekly beginning on day 0 and prior to both the first and second vaccination. GnRH antibody titers were determined using an ELISA assay. Serum testosterone levels were determined using radioimmunoassay. Semen samples were collected on day 0, prior to being vaccinated, and day 28 and evaluated for volume, percent motility, concentration, and morphology. Preliminary data reveals that the B-cell adjuvants did not improve the cGRFI’s ability to reduce fertility in male dogs. This study represents another step in the refinement of a one-shot contraceptive vaccine for the male dog.

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Group 1 - cGRFI Animals: O, N, H, E, and D Group 2 - cGRFI + Sigma Animals: M, L, K, I, and F Group 3 - cGRFI + Flagellin Animals: A, B, C, G, and J Group 4 - Placebo Animals: P, Q, R, S, and V

Until recently, Pfizer produced a commercially available vaccine (cGRFI) aimed at treating benign prostatic hyperplasia in canines. Although this vaccine has not been specifically Figure 1: The hypothalamictested in controlling fertility, it decreased serum pituitary-gonadal axis cascade will GnRH and reduced testosterone levels in be interrupted if GnRH antibodies are formed. treated dogs. This vaccine shows promise in inducing immunocastration in male dogs as an alternative to surgical intervention. Furthermore, the B-cell adjuvants, flagellin and Sigma Adjuvant System, have shown the ability to stimulate a strong antibody response and could act to increase GnRH titers induced by cGRFI (Cuadros, 2004).


Materials and Methods

Treatment Groups: The experiment consisted of 20 intact male dogs, of various breeds, between the ages of eighteen months and five years. Dogs were assigned to one of four treatment groups. All vaccinations were given subcutaneously in the right pelvic limb above the knee.

Serum Testosterone Levels

Change in Mean Semen Volume over Four Weeks

First Vaccination 6/16/2009

Booster 6/30/2009

Pfizer cGRFI 
None 1.0 ml 
Pfizer cGRFI 
Sigma Adjuvant 1.0 ml Pfizer cGRFI 
Flagellin 1.0 ml 
0.05 ml Saline 
None 1.0 ml 


Pfizer cGRFI 1.0 ml Saline 1.0 ml Saline 1.0 ml Saline 1.0 ml

Treatment Group

Manual Semen Collection and Evaluation: Semen samples were collected from each dog on day 0 and day 28. Collection involved digital stimulation and massage through the prepuce at the level of the bulbus glandis (Kutzler, 2005). Volume, color, concentration, motility, and morphology were recorded. Semen were classified as normal or abnormal with primary abnormalities (occurring during spermatogenesis) or secondary abnormalities (occurring during epididymal storage).

3 2 1 0 cGRFI

cGRFI + Sigma Adjuvant

Volume on 6/16/09

cGRFI + Flagellin

Placebo

e

Change in Mean Sperm Morphology over Four Weeks

h

i

f

d



On day 0 (6/16/09), animal D had prostatitis and provided an azoospermic sample. On day 28 (7/14/09), animals D, F, N, and O, provided a sample, however there was not enough volume to perform evaluation of motility, concentration or morphology. Table 2: Mean change in semen characteristics from day 0 to day 28. Only the cGRFI group showed a statistically significant decrease in motility (p value < 0.05). Volume decrease and concentration increase were positively correlated (see figure 3). The B-cell adjuvant vaccination groups did not improve the ability of the cGRFI vaccine to decrease fertility.

Placebo

14.2

29.2

2

2.2

23.8

27.2

72.8

66.25

71.75

67.6

38.5

cGRFI cGRFI (6/16/09) (7/14/09)

74.2 55

70.6

cGRFI + cGRFI + cGRFI + cGRFI + Placebo Placebo Sigma Sigma Flagellin Flagellin (6/16/09) (7/14/09) Adjuvant Adjuvant (6/16/09) (7/14/09) (6/16/09) (7/14/09) Treatment Group

Normal Sperm (%)

Primary Abnormalities (%)

Secondary Abnormalities (%)

Vaccination Reactions

Semen Evaluation:

cGRFI + Sigma Adjuvant cGRFI + Flagellin

25.75

3

Figure 4: Although there was a considerable decrease in percent normal sperm in the cGRFI group, it was not statistically significant (p value = 0.22).

Results

cGRFI

2.5

26.2

43.5

Jugular Venipuncture: 10 ml of blood was collected weekly every Tuesday between 8 and 11 AM over six consecutive weeks, beginning on day 0, without the use of anesthetic (per IACUC protocol). Serum was used to determine the following:

Serum testosterone concentrations using radioimmunoassay (Coat-A-Count, Siemens)

GnRH antibody titers using an ELISA assay

Treatment Group

1

18

30.8

g

Figure 2: Examples of sperm morphology: (a) proximal droplet, (b) tightly coiled tail, (c) distal droplet, (d) detached head, (e) normal, (f) 90 degree midpiece, (g) 90 degree head, (h) reflex midpiece with 90 degree head, and (i) stain artifact (tail curled back towards head). Primary abnormalities include a, b, f, g, and h. Secondary abnormalities include c and d. An eosin-nigrosin stain was used in the above slides.

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2.75 31

c

Volume on 7/14/09

Figure 3: All treatment groups except the placebo showed a statistically significant decrease in semen volume (p value