John Verstegen

Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste.

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.

Risk factors and reproductive disorders associated with canine herpesvirus-1 (CHV-1)

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with fertility disorders and neonatal mortality. In this study we screened for risk factors affecting CHV-1 antibody titers and investigated the association between antibody titers and reproductive disorders. Therefore, serum from 545 dogs used for reproduction was analysed with an ELISA. Using a forward stepwise procedure and retaining significant risk factors (P<0.05), best fitting multifactorial generalized linear model (glm) procedures were built for males and females. The effect of antibody titers on reproductive disorders was analysed with logistic regression analysis. The association between reproductive disorders and seroprevalence was analysed in chi-square analyses using contingency tables. In both sexes, kennel cough and breeding management were found to have an impact on the CHV-1 antibody titer. Also, the influence of kennel cough on the antibody titer was correlated to the hygienic status of the kennel. In females, age, kennel size and cycle stage had an effect on CHV-1 antibody titers. Furthermore, kennel size and hygiene were found to be correlated. In males, mating experience had an impact on CHV-1 antibody titers. An association was observed between serological status and a history of abortion in bitches. In conclusion, this study suggests CHV-1 antibody titers may be affected by many factors, both on an environmental and host level. Therefore, interpretation of the serological status requires precaution. Furthermore, oronasal and venereal transmission seem to play a role in the spreading of infection.

Laparoscopic Intrauterine Insemination in the Bitch

A technique for laparoscopic intrauterine insemination in bitches is described. During natural estrus, 5 beagle bitches were inseminated and S others were naturally mated (control group) twice at a 48-h interval on Days 3 and S (n = 4) or Days 4 and 6 (n = 6) after the increase in plasma progesterone considered to be indicative of the day of the preovulatory LH peak. All the inseminations were with fresh semen and under general anesthesia. The technique involved the introductions of 1) a Verres needle to insufflate the abdominal cavity by direct punction on the middle line 1 cm over the umbilicus, 2) a laparoscope to visualize the abdominal cavity by a 1 cm puncture on the middle line 1 cm under the umbilicus, 3) a forceps used to manipulate the uterus by a 0.5 cm puncture at 2 to 3 cm lateral to the mammary glands, and 4) an 18-g catheter used to puncture the uterus on the middle line between the 3rd and 5th mammary gland. The uterine body was grasped by the forceps and elevated against the ventral abdominal wall. The 18-g catheter was then inserted through the abdominal wall directly into the uterine lumen, and 1.0 ml of fresh semen containing 250 to 480 x 10(6) spermatozoa/ml was injected. The inseminations resulted in pregnancies in all animals. Litter size was similar in the artificially inseminated and naturally mated bitches (5 +/- 1.8 and 4.8 +/- 1.6 pups per litter, respectively). Bitches in the artificially inseminated group delivered at 65.2 +/- 0.8 d and in the natural mated group at 65.4 +/- 0.5 d after the LH peak. In conclusion, this paper gives the first results of intrauterine laparoscopic insemination in bitches, indicating interesting perspectives for this technique in dogs reproduction.

Changes in oestrogen, progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus.The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.

Relationship between receptors for epidermal growth factor and steroid hormones in normal, dysplastic and neoplastic canine mammary tissues

The concentrations of receptors for epidermal growth factor (EGF-R), oestrogen (ER) and progesterone (PR) were measured in 108 samples from canine mammary tumours and 132 samples of normal mammary tissue removed surgically from 84 bitches. The history and clinical signs were also recorded. Binding sites of high affinity were detected in 70 per cent of both types of tissue and no significant variations in EGF-R concentrations or positivity were observed with the histology, location, size or number of mammary tumours or the age of the animal. A significant direct correlation (P = 0.002) was observed between the concentrations of ER and EGF-R only in malignant tumours. The concentrations of EGF-R were significantly correlated (P = 0.04) in normal mammary tissues adjacent to and distant from the lesions, but not between normal tissue and tumour tissue. No significant differences were observed in the expression of EGF-R in normal and neoplastic tissues from the same bitches. The direct correlation between the concentrations of EGF-R and ER in malignant tumours could be related to an oestrogen-dependent expression of EGF-R or to a similar pattern of regulation of the receptors.

Effects of a medetomidine‐midazolam‐butorphanol combination on renal cortical, intestinal and muscle microvascular blood flow in isoflurane anaesthetized dogs: a laser Doppler study

OBJECTIVE To obtain renal cortical, ileal, colonic and skeletal muscle microvascular blood flow measurements in dogs using the laser Doppler technique and to characterize the effects of medetomidine-midazolam-butorphanol combination on these flows. STUDY DESIGN Prospective randomized experimental study. ANIMALS Fourteen clinically normal beagles (two groups of seven), aged 1-4 years and weighing 13.2 ± 1.8 kg. METHODS All dogs were anaesthetized with 1.7% end-tidal isoflurane in oxygen. In the treatment group, after instrumentation and when anaesthesia was considered stable, medetomidine (1 mg m-2 body surface area (BSA)) was administered intramuscularly (time 0). Midazolam (1 mg kg-1) and butorphanol (0.1 mg kg-1) were administered intravenously 20 minutes later. Atipamezole (2.5 mg m-2 BSA) was administered intramuscularly 60 minutes after medetomidine. In the control group, saline (0.5, 2.5 and 0.25 mL) was administered at the corresponding times. Heart rate, systolic, diastolic and mean arterial pressures, body temperature, renal cortical, ileal, colonic and skeletal muscle microvascular blood flows were measured before time 0, and 5, 15, 25, 40, 60, 65, 70 and 90 minutes thereafter. RESULTS Heart rate, ileal and skeletal muscle blood flows decreased in the treatment group, while no changes were observed in the control group. CONCLUSIONS Laser Doppler flowmetry allowed the measurement of microvascular blood flow in different organs. The medetomidine-midazolam-butorphanol combination decreases intestinal and skeletal muscle microvascular blood flows, while renal cortical blood flow is maintained. CLINICAL RELEVANCE Medetomidine-midazolam-butorphanol combination can be used to induce a short duration anaesthesia in dogs, but it will induce cardiovascular depression. This depression appears to be accompanied by a redistribution of blood flow.

Radiographic contrast medium for uterine insemination in the bitch, and its effect upon the quality and fertility of fresh dog semen

Four ejaculates were collected from each of 6 adult male beagle dogs. The second fraction was divided into 3 aliquants which were then diluted with physiological saline, an isoosmolar solution of sodium/meglumine diatroate, and an iso-osmolar solution of iohexol. The diluted samples were incubated at 39 degrees C and evaluated at 0, 60, 90, 120, 240 and 360 min after dilution. A variety of assessments was made, including, spermatozoal motility, spermatozoal morphology, and acrosorne status. The practicality of using 1.0 ml of iso-osmolar contrast medium combined with radiographic examination was evaluated as a method of confirming accurate placement of a transcervical uterine catheter by injecting contrast after positioning the catheter in 4 beagle bitches. The effect of the procedure on fertility was assessed using 5 greyhound bitches which were inseminated with fresh semen and in which pregnancy was monitored using diagnostic B-mode ultrasound imaging. There was no significant difference between physiological saline and the sodium/meglumine diatroate solution upon semen quality, while the iohexol solution produced a significant reduction in spermatozoal motility and morphology. No adverse clinical effects were observed when contrast medium was administered into the uterus to either group of bitches. A subjective assessment of radiographic quality showed that the sodium/meglumine diatroate solution, which contained twice the iodine concentration of the iohexol solution, produced significantly greater radiopacity and was radiographically more useful than the iohexol solution. The sodium/meglurnine diatroate solution had no adverse effect upon the fertility of dog semen, and all bitches that were inseminated with this technique conceived and maintained the pregnancy to term. Litter size was considered to be normal for the breed. Small volumes of an iso-osmolar solution of sodium/meglumine diatroate may be useful for ensuring correct placement of transcervical catheters prior to artificial insemination in the bitch.