Effect of administrating oxytocin or prostaglandin F_2α on characteristics of the canine ejaculate

Abstract

To test the hypothesis that male dogs treated with smooth muscle contracting drugs have an increase in the total number of spermatozoa in the ejaculate but no change in all other ejaculate characteristics, such as progressive motility of spermatozoa or percentage morphologically normal spermatozoa, dogs were treated with oxytocin or prostaglandin F_2α (PGF_2α) and compared to saline treatments. Semen was collected from each of the 3 dogs once every 3 to 4 d for a total of 6 collections per dog. Ten minutes before each collection, 1 of 3 injections (oxytocin 10 IU [0.5 mL], IM; PGF_2α 2.5 mg [0.5 mL], IM; or saline 0.5 mL, IM) was administered. Compared to the saline controls, neither treatment had any significant effect on any measured variable when collected in this manner with an estrus bitch present. Therefore, the use of these drugs does not appear to be a viable treatment to increase the number of spermatozoa.

Introduction

Bitches must be inseminated with at least 250 million morphologically normal spermatozoa over the fertile period of the estrus cycle for pregnancy to occur reliably (1). This number can be reduced by intrauterine insemination. However, if the preferred stud dog is subfertile, a large number of collections and breedings may still be required but rendered impossible because of the variability in length of the estrus cycle, distance considerations, or temperament incompatibilities. If an agent could be found that would reliably increase the total number of spermatozoa per ejaculate by depleting extragonadal reserves, the quality of semen samples used for artificial insemination would be improved in cases where semen quality was less than desired.

In normal antegrade ejaculation in the dog, 3 sequential processes occur: seminal emission, bladder neck closure, and seminal expulsion through the penile urethra. Seminal emission occurs when sympathetic stimulation of the epididymis and vas deferens causes flow of spermatozoa and seminal fluid into the prostatic urethra (2–5). Sympathetic stimulation also causes partial closure of the bladder neck. This leads to the formation of a pressure chamber within the urethra, which signals the firing of sympathetic fibers to stimulate complete closure of the bladder neck and contractions of the prostate. Finally, somatic nerves are stimulated to begin clonic contractions of the striated muscles of the penis. Waves of contractions occur along the bulbocavernosus and ischiocavernosus muscles, which encircle the entire length of the penile urethra, leading to seminal expulsion (3–6). Numerous pharmacological agents have been shown to enhance male reproductive performance in many species. (7–13)

The purpose of this project was to examine the influence of 2 examples (oxytocin and prostaglandin F_2α [PGF_2α]) of 1 class of drug, a smooth muscle stimulant, on the volume, total number of spermatozoa in the ejaculate, percentage progressive motility of spermatozoa, and percentage of morphologically normal spermatozoa in the dog.

Materials and methods

Three client-owned, intact male Samoyeds were used in the study. All dogs were given a complete physical and reproductive examination, including transrectal palpation of the prostate, palpation of the testes, and measurements of scrotal width. A complete semen evaluation was performed on the ejaculate, including notation of the overall color and opacity of the sample, total count of spermatozoa, notation of the number of progressively motile spermatozoa, measure of total volume, evaluation of normal and abnormal spermatozoa, and calculation of the percentage of normal and progressively motile normal spermatozoa. The ejaculate was cultured for aerobic bacteria, anaerobic bacteria, and Mycoplasma spp., and cytologic examination of the seminal fluid was performed. These cultures were done knowing that any significant positive results would necessitate all dogs being placed on an appropriate antibiotic before the study began and for the duration of the study. Antibiotics would be given to reduce the confounding factors that an infection might have on the study and because of the uncertainty of knowing if a positive culture reflected true infection or normal flora. Significant organisms were defined as any aerobic organisms at a concentration of greater than 10⁴ colony-forming units (CFU)/mL, or any anaerobic organism. Mycoplasmas were not considered significant, as they are often present in normal dogs and have not been shown to be definitively pathogenic when cultured from the reproductive tract of male dogs (unpublished observations), Serologic testing for antibodies to Brucella canis was done by using a rapid card agglutination test.

Collection method

Semen was collected from each dog by an experienced operator using a latex artificial vagina (AV) and manual stimulation once every 3 to 4 d for a total of 6 collections. All dogs had had experience with collection and none mounted the bitch in estrus that was present at all of the collections. Each collection ended when prostatic fluid had been flowing long enough for the operator to be fairly certain that a full sperm rich portion had been obtained. The total volume collected was measured and portions were not separated.

The dogs were treated with 1 of 3 injections 10 min before each collection. Treatments were either oxytocin (Oxytocin Injection; Vedco, St. Joseph, Missouri, USA), 10 IU (0.5 mL), IM; PGF_2α (Lutalyse; Upjohn, Kalamazoo, Michigan, USA), 2500 μg (0.5 mL), IM; or sterile saline, 0.5 mL, IM. Each dog received each of the above 3 treatments 2 × for a total of 6 treatments; therefore, there was a total of 18 collections. The sterile saline injections allowed each dog to act as his own control. An independent observer randomly labeled all injections for each dog, so the injections for dog A were labeled 1 to 6A; for dog B, 1 to 6B; and for dog C, 1 to 6C; thus, the operators were blinded to which injections they were giving at each collection. All injections were also given in the same manner (IM) so as to keep the identity of the treatment or placebo hidden from the operator.

Semen evaluation

Semen was assessed immediately for its volume and percentage of progressively motile spermatozoa. It was evaluated within 1 h of collection, for total number of spermatozoa in the ejaculate and the percentage of morphologically normal spermatozoa (14).

Statistics

Statistical evaluation was done by randomized complete block analysis of variance (ANOVA). Significant difference was acknowledged if P < 0.05. The hypothesis to be tested was that there was no change in the progressive motility of spermatozoa or the percentage of morphologically normal spermatozoa, but an increase in total number of spermatozoa, in the ejaculate, when dogs were treated with oxytocin or PGF_2α compared with sterile saline.

Results

Physical examination and semen culture

On initial physical examination, dogs A and B appeared to be healthy. Dog C had a draining mass in the lower right perianal area. An impression smear done on the mass revealed healthy neutrophils and normal noncornified epithelial cells. This lesion was thought to be associated with the anal gland and it resolved over the course of the study with regular cleaning and antibiotic therapy, as described later. Dog C also had asymmetrical testes, with the smaller testis softer than the other. All dogs had a normal prostate, and scrotal width measurements were within normal limits.

Results of semen cultures revealed that dog A had a positive anaerobic culture (3+ Bacteroides sp), a positive aerobic culture (greater than 10⁶ beta hemolytic Escherichia coli and beta hemolytic Streptococcus sp.), and positive mycoplasma cultures. Dog B had a positive mycoplasma culture. Dog C had positive aerobic (greater than 10⁶ E. coli (beta-hem) and Proteus mirabilis and positive mycoplasma cultures. Serologic testing for antibody to Brucella canis was negative for all dogs. Seminal fluid was interpreted as being normal in all 3 dogs on cytologic cytologic examination. Because the dogs had positive culture results, all dogs were administered ampicillin (Bristol Myers, New York, New York, USA), 500 mg, PO, q8h, for the duration of the study.

Treatment semen evaluations

The semen evaluations of the 3 Samoyed stud dogs were analyzed and recorded (Figure 1). Data for all dogs include 2 samples in each data point, except for dog A in which only 1 sample was obtained for PGF_2α and saline because no ejaculate was obtained after the last 2 treatments.

Figure 1.

Characteristics of semen evaluations performed on 3 treated Samoyed dogs.

The mean volume of semen for each dog showed no differences between treatments of oxytocin, PGF_2α, and saline. The difference between dogs was significant when volume produced was measured. Similarly, mean progressive motility of spermatozoa among treatments was similar, while dog with dog comparison was significantly different. The mean number of spermatozoa was unchanged when treatments with either oxytocin or PGF_2α were compared with saline. The difference between dogs when measuring this variable was significant. The dog also had a significant effect on the percentage of normal spermatozoa in the ejaculate. The treatments given did not make any significant difference (Figure 1).

No side effects, such as panting or salivating, which sometimes are associated with PGF_2α treatment, were noted after administration of either drug.

Discussion

Smooth muscle contracting drugs do not appear to have an effect when used in the manner of this study. However, further study is needed using other techniques and environments before the use of smooth muscle contracting drugs is ruled out as ineffective. For example, it is not known if smooth muscle in the male canine reproductive tract contains receptors for PGF_2α, oxytocin, or both. It has been shown that receptors for these drugs exist in the reproductive tract of the male mouse and boar (15,16). It is known that semen quality is highly variable between dogs and even within the same dog at different collection times (unpublished observations), the natural variability between collections may have accounted for our not being able to establish conclusive evidence of the effects of smooth muscle contracting drugs on canine ejaculation.

Ampicillin treatment was provided in order to eliminate the confounding factors an infection might cause this study, even though the authors are aware that these organisms may be normal flora. Dogs B and C received antibiotics only to maintain consistency between treated dogs. Ampicillin was chosen because it is one of the drugs that has the ability to penetrate the prostate and because anaerobic and aerobic bacteria cultured proved sensitive to this drug.

The presence of a bitch in estrus may influence semen quality (17,18). It is possible that the presence of the bitch in estrus may have influenced the results in this study, in that maximal output may have already been reached.

In order to completely understand the potential benefit that smooth muscle contracting drugs may have on increasing male spermatozoa number and increased fertilization ability of stud dogs, the presence or absence of PGF_2α and oxytocin receptors in the canine reproductive tract needs to be established and a larger group of dogs studied.

An alternative study design would be to collect semen from dogs once daily after treatment until the number of spermatozoa in the ejaculate stabilized, reflecting the daily sperm output (DSO). A comparison of number of days to reach DSO between treatments might better demonstrate effect of drug therapy. A significant decrease in the number of days required to reach DSO would show that the treatments were having a positive effect on the number of spermatozoa in each ejaculate.