K. Onclin

Computer assisted semen analyzers in andrology research and veterinary practice

The evaluation of sperm cell motility and morphology is an essential parameter in the examination of sperm quality and in the establishment of correlations between sperm quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective assessment of different cell characteristics: motion, velocity, and morphology. The development and problems related to this technology are raised in this review, paying particular attention to the biases and standardization requirements absolutely needed to obtain useful results. Although some interesting results, mainly in humans, have already been obtained, many questions remain, which have to be answered to allow for further development of this technology in veterinary medicine, clinical fertility settings, physiological, and toxicology research activities. The main problem is related to the standardization and optimization of the equipment and procedures. The different CASA instruments have all demonstrated high levels of precision and reliability using different sperm classification methodology. Their availability gives us a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa.

Effect of stage of anestrus on the induction of estrus by the dopamine agonist cabergoline in dogs

Beagle bitches were administered the dopamine D2 receptor agonist cabergoline in 3 groups of 5 animals each, starting on known days of the estrous cycle. Cabergoline treatment was started in either early anestrus (Days 93 to 108), mid-anestrus (Days 123 to 156), or late anestrus (Days 161 to 192) at doses of 5 ug/kg/d, per os, and was continued until the confirmation of induced proestrus or for 40 d. Reproductive parameters were compared with those in 5 control anestrous bitches (Days 90 to 150). In control bitches, the mean (+/- SEM) interval to the next proestrus (73+/-11 d) resulted in an interestrus interval (192+/-9 d) similar to that of the previous cycles (196+/-11 d). In 14 of the 15 cabergoline-treated bitches, the next proestrus occurred within 4 to 30 d, was premature in early and mid-anestrous bitches and developed with low variability within groups. The resulting intervals to proestrus in bitches treated with cabergoline in early anestrus (20+/-2 d), mid-anestrus (14+/-3 d) and late anestrus (6+/-1 d) resulted in interestrus intervals in those groups of 131+/-5, 166+/-7 and 196+/-2 d, respectively. In response to treatment, interestrus intervals were reduced (P<0.05) and more synchronous (P<0.05) in early and mid-anestrus bitches, and were more synchronous (P<0.05) in late-anestrous bitches compared with those of control bitches or those of the previous cycle. Periovulatory estradiol and progesterone profiles of induced cycles in treated bitches were similar to those of spontaneous cycles in control bitches. Four of 5 control bitches and 12 of the 14 responding cabergoline-treated bitches became pregnant and produced normal litters. Plasma prolactin concentrations at Days 2 and 5 of treatment (0.3+/-0.1 ng/mL) and at the onset of proestrus shortly before the end of treatment (0.4+/-0.1 ng/mL) were lower (P<0.05) than those present in anestrus prior to treatment (1.7+/-0.6 ng/mL) or in control bitches. Prolactin was also low at the onset of proestrus in control bitches (0.5+/-0.2 ng/mL). The results demonstrate that prolactin-lowering doses of the dopamine agonist cabergoline can terminate the normal obligate anestrus in dogs, and that the effect occurs more slowly in early anestrus than in mid or late anestrus.

Comparisons of intravaginal and intrauterine insemination of bitches with fresh or frozen semen

To compare the importance of the route of insemination when using fresh or frozen semen, six groups of five bitches were inseminated either into the uterus (groups 4, 5 and 6) or the vagina (groups 1, 2 and 3) with fresh (groups 1 and 4) or frozen semen (groups 2, 3, 5 and 6). The fresh semen was collected when needed from the same dog. The frozen semen used in groups 2 and 5 was obtained from seven dogs on the same day, and pooled and processed simultaneously so that the groups were inseminated with exactly the same semen. The frozen semen used in groups 3 and 6 was obtained from different dogs and processed independently to evaluate not only the effect of the route of insemination but also the potential effect of the dog. The mean concentration of the fresh semen was 310 x 106 spermatozoa/ml, its motility was greater than 80 per cent and the percentage of normal live spermatozoa was 80 to 92 per cent. The mean spermatozoal concentration of the frozen semen was 200 x 106 spermatozoa/ml, its motility was greater than 60 per cent and the percentage of normal live spermatozoa was 80 per cent. In all the groups there were fewer than 15 per cent abnormal spermatozoa. The animals inseminated with fresh semen received significantly more spermatozoa than the others. The bitches were inseminated twice, three and five days after the estimated peak of luteinising hormone, with a total volume of 5 ml for the vaginal inseminations and 2 ml for the intrauterine inseminations. Sixty per cent of the bitches inseminated with frozen semen and 100 per cent of the bitches inseminated with fresh semen became pregnant, irrespective of the insemination technique used.

Comparisons of estradiol, LH and FSH patterns in pregnant and nonpregnant beagle bitches.

To characterize plasma estradiol, LH and FSH patterns of secretion during the bitch estrous cycle, blood samples were obtained daily from 15 days before until 135 days after the LH surge in 10 pregnant and 10 nonpregnant beagle bitches. After an initial increase between days 15 and 10 and an expected proestrous peak, estradiol concentrations increased again from days 9-12 (corresponding to cytological metestrus) from basal values observed around day 9 after the LH surge, and remained significantly elevated throughout the luteal phase both in pregnant and nonpregnant animals. Concomitantly with the end of the luteal phase, plasma concentrations of estradiol returned to basal values in both groups. During the mid- to late-luteal phase, mean basal LH secretion was significantly elevated throughout in the pregnant relative to the nonpregnant animals. However, in nonpregnant animals, pulsatility was increased and peaks of higher amplitude were observed. The plasma FSH profiles, determined by a specific homologous RIA, differed significantly between pregnant and nonpregnant bitches during the last two-thirds of the luteal phase with a mean FSH level more elevated during pregnancy. The FSH level then decreased around parturition and low concentrations during lactation period were observed. The FSH concentrations remained steady in nonpregnant luteal phases from early luteal phase through mid-anestrus. The differences in pregnant and nonpregnant LH and FSH concentrations suggest pregnancy differences in regulation of the corpus luteum. Finally, the elevated estradiol concentrations observed during the luteal phase of both pregnant and nonpregnant animals suggest that an ovarian production of estrogens may be involved in overall corpus luteum regulation in dogs as in other species.

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.

Comparisons of different combinations of analogues of PGF2α and dopamine agonists for the termination of pregnancy in dogs

Groups of five pregnant bitches were treated to terminate the pregnancy with four combinations of drugs, starting 28 days after the estimated surge of luteinising hormone (LH), 22 to 28 days after the first mating. The treatments were: cabergoline administered orally for 10 days at a dose of 5 μg/kg and a single subcutaneous injection of 2.5 μg/kg cloprostenol at the start of the treatment; the same dose of cabergoline plus two doses of 1 μg/kg cloprostenol administered on days 28 and 32 after the LH surge; bromocryptine administered orally at a dose of 30 μg/kg three times a day for 10 days plus a single dose of 2.5 μg/kg cloprostenol; the same dose of bromocryptine plus two doses of 1 μg/kg cloprostenol; and a group of five pregnant bitches was left untreated. The pregnancies were terminated in all but one of the treated bitches, in each case by resorption of the fetuses. There were few side effects in the bitches treated with two doses of 1 μg/kg cloprostenol, and were present but acceptable in those treated with one dose of 2.5 μg/kg. Plasma progesterone concentrations decreased to less than 1 ng/ml within 72 hours of the start of treatment and remained low except in the bitch in which pregnancy was not terminated. In the five untreated bitches, plasma progesterone remained high and they whelped normally. In the treated groups, the intervals between successive displays of oestrus were reduced by approximately 70 days in comparison with previous cycles or with the control group, but the fertility of the dogs was not affected adversely.

Termination of unwanted pregnancy in dogs with the dopamine agonist, cabergoline, in combination with a synthetic analog of PGF2alpha, either cloprostenol or alphaprostol

Associations of a dopamine agonist, cabergoline, and a synthetic analog of PGF2alpha, either alphaprostol or cloprostenol, were used to induce pregnancy termination in 15 mature Beagle bitches. Alphaprostol (20 microg/kg/d) was injected subcutaneously (sc) in combination with cabergoline (1.65 microg/kg/d, sc) daily for 5 d from Day 32 after the LH peak in 5 animals. Cloprostenol 2.5 microg/kg/d in one group (n = 5) and 1 microg/kg/d in another group (n = 5) was also used in combination with the same dosage of cabergoline, following the same protocol, but starting from Day 25 after the LH peak. Treatment efficacy, defined by the number of pregnancy terminations, was 100% in all treated groups. Dose-related side effects due to PGF2alpha were observed (excessive salivation, prostration, vomiting, diarrhoea, scratching at the injection site), and were less severe with cloprostenol than with alphaprostol. In the group treated with the lowest dosage of cloprostenol, no adverse reactions were noticed. In all treated groups, interestrus intervals compared with previous cycles and in control animals were reduced after treatment.