The vascular network within the developing mammary gland (MG) grows in concert with the epithelium to prepare for lactation, although the mechanisms coordinating this vascular development are unresolved. Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and vascular permeability in the MG during pregnancy and lactation, where its expression is upregulated by prolactin. Given our previous finding that late-gestational hyperprolactinemia induced by domperidone (DOM) increased subsequent milk yield from gilts, we sought to establish changes in vascular development during late gestation and lactation in the MGs of these pigs and determine whether DOM altered MG angiogenesis and the factors regulating it. Gilts received either no treatment (n = 6) or DOM (n = 6) during late gestation, then had their MG biopsied from late gestation through lactation to assess microvessel density, VEGF-A distribution and messenger RNA expression, and aquaporin (AQP) gene expression. Microvessel density in the MG was unchanged during gestation then increased between days 2 and 21 of lactation (P < 0.05). The local expression of messenger RNA for VEGF-A120, VEGF-A147, VEGF-A164, VEGF-A164b, VEGF-A188, VEGF receptors-1 and -2, and AQP1 and AQP3 all generally increased during the transition from gestation to lactation (P < 0.05). Immunostaining localized VEGF-A to the apical cytoplasm of secretory epithelial cells, consistent with a far greater concentration of VEGF-A in colostrum and/or milk vs plasma (P < 0.0001). There was no effect of DOM on any of the variables analyzed. In summary, we found that vascular development in the MG increases during lactation in first-parity gilts and that VEGF-A is a part of the mammary secretome. Although late-gestational hyperprolactinemia increases milk yield, there was no evidence that it altered vascular development.

Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5 × 1011 virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2 × 1012 vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems.

The voluntary consumption of alcohol by humans is a modifiable lifestyle factor that has been consistently linked to a woman's risk of developing breast cancer. We have used an animal model that closely recapitulates breast development in humans to study the effect of alcohol intake on breast growth and morphology. Pubertal female pigs were fed alcohol for 4-5 weeks at 19-21% of total caloric intake, which led to average blood alcohol concentrations of 115-130mg/dL. Alongside increased liver mass, alcohol intake promoted the formation of distended ductules within lobular units in association with increased epithelial proliferation. Alcohol consumption also increased phosphorylation of the transcription factor STAT5 in the mammary epithelium, but did not lead to any evidence of precocious lactogenesis. In conclusion, feeding alcohol to female pigs having a similar physiology and mammary gland morphology to humans during a reproductive state equivalent to human adolescence leads to increased mammary gland proliferation and development of atypical lobular structures. These changes may phenocopy how alcohol intake increases the risk for developing breast cancer in humans.

We have investigated GnRH immunization for the treatment of urethral sphincter mechanism incompetence in ovariectomized bitches. It has been reported that decreasing LH secretion through the use of GnRH agonists temporarily restores continence in some bitches. Therefore, decreasing the circulating LH concentrations by immunizing against GnRH might temporarily maintain continence in incontinent dogs. Sixteen incontinent dogs given phenylpropanolamine (PPA) to control incontinence were recruited for this study. Eleven dogs were immunized against GnRH (novel treatment group) at week 0, and nine dogs were vaccinated again 4 weeks later. Five dogs (standard treatment group) were vaccinated with a placebo twice at 4-week intervals. PPA was discontinued in the novel treatment group 2 weeks after revaccination, and standard-treatment dogs were given PPA for the duration of the study. Blood samples were collected before each treatment and at 6, 8, 10, 12, 16, 20, and 24 weeks and owners recorded episodes of incontinence throughout the study. Ten of the eleven dogs in the novel treatment group experienced side effects as a result of vaccination; two of these dogs experienced more severe side effects after the first vaccination and were withdrawn from the study as a result. Of the nine dogs that completed the vaccination series, four dogs remained continent after PPA was discontinued. For these four dogs, there was no difference in incontinent episodes when they were given PPA versus treatment with the vaccine. All nine novel-treatment dogs developed a GnRH antibody titer and experienced a significant decrease in circulating LH concentrations. In conclusion, GnRH immunization was effective in maintaining continence in four of the nine incontinent ovariectomized dogs, and in these dogs, treatment with the vaccine was comparable with treatment with PPA.

Reproductive function in the dog is controlled by feedback mechanisms that involve the hypothalamus, the anterior pituitary gland, and the gonads. Surgical gonad removal, a common procedure performed in dogs for the purposes of sterilization, disrupts the hypothalamic-pituitary-gonadal (HPG) axis and results in permanently elevated concentrations of gonadotropins. Manipulation of the HPG axis via gonadotropin releasing hormone (GnRH) immunization results in the synthesis of GnRH neutralizing antibodies, which bind to (neutralize) GnRH and prevent it from binding to its receptors. The end result of GnRH immunization is the cessation of pituitary gonadotropin secretion, namely luteinizing hormone (LH).In 2004, a commercial GnRH vaccine was launched in the United States (Canine Gonadotropin Releasing Factor Immunotherapeutic®; Pfizer Animal Health USA), labeled for the treatment of benign prostatic hyperplasia in intact male dogs. This research investigated two novel clinical applications of this vaccine in dogs. In the first study, physiologic responses to GnRH immunization in intact male dogs were observed. Four intact males were vaccinated with the GnRH vaccine twice at four week intervals. Blood samples were collected prior to each injection (at weeks 0 and 4) and at weeks 12 and 20 following initial vaccination. Scrotal measurements were also made at the time of each blood sample collection to calculate testicular volume. All four dogs developed a GnRH antibody titer and experienced a significant decrease in testosterone concentrations. Testicular volume also significantly decreased, and this effect was reversed by the end of the study. LH concentrations remained at basal levels. These results are indicative of temporary humoral response to the GnRH vaccine, and future studies should investigate prolonging these effects to potentiate GnRH immunization as a method of population control in dogs. Beyond use as a temporary immunosterilant, GnRH immunization has other promising clinical applications. Elevated gonadotropin concentrations as a result of gonad removal in female dogs decreases urethral pressure, and in some bitches, this results in the development of urethral sphincter mechanism incompetence (USMI). Therefore, lowering gonadotropin concentrations in incontinent ovariectomized bitches through GnRH immunization may restore continence. In the second study, sixteen incontinent dogs that were using phenylpropanolamine (PPA) to control incontinencewere recruited. Eleven dogs were immunized against GnRH at week 0, and nine dogs were vaccinated again four weeks later. Five control dogs were vaccinated with a placebo twice at four week intervals. Vaccinated dogs discontinued PPA two weeks after re-vaccination, and control dogs remained on PPA for the duration of the study. Blood samples were collected before each injection and at 6, 8, 10, 12, 16, 20, and 24 weeks, and owners recorded episodes of incontinence throughout the study. Of the nine dogs that completed the vaccination series, four dogs…

We have investigated the effect of immunization against gonadotropin releasing hormone (GnRH) using a commercial canine GnRH vaccine on testosterone concentration, testicular volume, testicular histology, and intermale behavior in intact male llamas and alpacas. Llamas (n=28) and alpacas (n=20) were either immunized (received 3 mL of vaccine given intramuscularly) or treated as controls (received 3 mL of sterile diluent given intramuscularly) at 0, 3, and 6 weeks. Blood samples and testicular volume measurements were taken at 0, 3, 6, 9, and 12 weeks. Owner surveys grading behavior at 0 and 12 weeks were received for 14 llamas. Two llamas at each time point undergoing the vaccination procedure were also castrated for testicular histological examination. Vaccinated animals elicited a GnRH antibody titer, and there was a significant decrease in testosterone concentration and testicular volume when compared with control animals. Intermale aggressive behavior was also significantly decreased in the surveyed llamas. However, histological examination revealed no significant changes. GnRH immunization using the canine GnRH vaccine may be an effective method for reducing intermale aggression in llamas and alpacas by decreasing circulating testosterone concentrations but cannot be recommended as an immunosterilant because of its lack of efficacy in interrupting spermatogenesis.

We investigated the effect of immunization against gonadotropin releasing hormone (GnRH) using a commercial canine GnRH vaccine on estrus suppression and unwanted estrous behavior in mares. In experiment 1, mares were immunized (n = 6) twice with vaccine (5 mL) given intramuscularly 4 weeks apart or received a control diluent (n = 5). Transrectal ultrasonographic examination of the reproductive tracts was performed three days a week for 40 weeks after initial vaccination. Blood samples were collected weekly for GnRH antibody titer and progesterone concentration determination. In experiment 2, privately-owned mares (n = 12) were immunized twice with vaccine (1 mL) given intramuscularly 4 weeks apart. Blood samples were collected prior to each vaccination as well as 12 and 20 weeks after initial treatment, and transrectal ultrasonographic examinations of the reproductive tracts were performed 12 weeks after the first vaccination. Vaccinated mares in experiment 1 responded with a GnRH antibody titer, progesterone concentrations significantly lower than controls, and cessation of ovarian activity. Vaccinated mares in experiment 2 also responded with a GnRH antibody titer, progesterone concentrations that remained basal for the duration of the study, and cessation of ovarian activity. Owners of vaccinated mares in experiment 2 reported that the number of unwanted estrous behaviors present before vaccination significantly decreased following vaccination. In conclusion, GnRH immunization using a canine GnRH vaccine is an effective method for suppressing estrus and unwanted estrous behavior.

We investigated the use of a commercial gonadotropin-releasing hormone (GnRH) vaccine as a method of temporary and reversible immunocastration in intact male dogs. Four privately owned dogs were vaccinated twice at 4-week intervals. Blood samples were collected at 0, 4, 12 and 20 weeks following the initial vaccination. These samples were analysed for GnRH antibody titres, luteinizing hormone (LH) and testosterone concentrations. Scrotal measurements were made at the time of sample collection, and testicular volume was calculated using the formula of an ellipsoid. As a result of vaccination, dogs displayed an elevated GnRH antibody titre, decreased LH and testosterone concentrations and decreased testicular volume, which reversed by the end of the study period. Therefore, these results suggest that immunizing against GnRH may be a possible choice for temporary and reversible immunocastration.