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I. K. M. Liu, M. Bernoco and M. Feldman

Summary. Ten fertile feral mares and 6 domestic horses (4 fertile mares, 1 infertile mare, 1 gelding) were immunized with heat-solubilized pig zonae pellucidae by 4 injections equivalent to 2000 or 5000 zonae each at 2–4-week intervals and a booster injection of 20 000 zonae 6–10 months after the last of the initial inoculations. The immune response was reflected by high antibody levels as measured by an enzyme-linked immunosorbent assay (ELISA) using immobilized pig zona antigen. In-vivo inhibition of fertility occurred in 12 (86%) of the 14 fertile mares studied and persisted for a minimum of 7 months. Repeated mating of the fertile domestic mares resulted in conception when anti-pig zona antibody concentrations had decreased from initial peak absorbance ratios (>1·0) to relatively lower levels (0·64 or less with one exception). An indirect immunofluorescence assay, revealed a considerably lower cross-reactive antibody titre with horse oocytes as compared to pig oocytes. Clinical, endocrinological and histological analyses of the ovaries and their function following regained fertility after immunization revealed no abnormalities. One mare remained infertile.

Keywords: horse; contraceptive vaccine; zona pellucida; antibodies; heteroimmunization.

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M. H. T. Troedsson, I. K. M. Liu, M. Ing, J. Pascoe and M. Thurmond

The electrical myometrial activity of three mares with a documented increased susceptibility to chronic uterine infection (CUI) and three mares considered to be resistant to CUI was investigated. Electrodes were surgically implanted in the myometrium of the mares and electrical activity was monitored by a Grass polygraph. Oestrus was determined by transrectal ultrasonography of the reproductive tract and teasing of the mares with a stallion. Findings were confirmed by blood progesterone concentrations <0.1 ng ml−1. At the third day of oestrus or when a follicle > 35 mm was detected, the uterus was infused with a genital strain of 5 × 106 Streptococcus zooepidemicus. Myometrial electrical activity was monitored for 1–4 h before the bacterial infusion and continued until a visual stabilization of the activity occurred. No statistically significant differences in electrical myometrial activity were detected between susceptible and resistant mares before the infusion of bacteria into the uterus. A visible increase in myometrial electrical activity was seen in all mares following the bacterial infusion. However, the myometrial response of susceptible and resistant mares was different. Resistant mares demonstrated a greater myometrial activity (P < 0.001) than did susceptible mares. These differences were observed in frequency (P < 0.005) as well as duration (P < 0.001) and intensity (P < 0.001) of the uterine activity. Differences were most marked between 10 and 20 h after the intrauterine inoculation of bacteria. It was concluded from this study that myometrial activity is an important part of the uterine defence mechanism in mares. Mares susceptible to CUI appear to have an impaired electrical myometrial activity in response to an intrauterine bacterial challenge.

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A. C. Enders, K. C. Lantz, I. K. M. Liu and S. Schlafke

Summary. Twelve blastocysts, collected 7–12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to > 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cells, forming an irregular layer of cells capping the epiblast. With continued increase in blastocyst size, polar trophoblast cells became isolated but maintained their characteristic apical endocytic structures. By Days 10–12, the scattered trophoblast cells showed evidence of deterioration, and vacuoles containing cell debris were common within the epiblast.

It is suggested that polar trophoblast cells become scattered, rather than withdrawing as a unit, because they become more adherent to subjacent epiblast cells than to adjacent trophoblast cells. It is further suggested that most of the isolated cells are eventually phagocytosed by epiblast cells.

Keywords: horse; blastocyst; trophoblast; differentiation; inner cell mass

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M. H. T. Troedsson, A. O. G. Wiström, I. K. M. Liu, M. Ing, J. Pascoe and M. Thurmond

A method for interpreting and analysing electromyographic (EMG) data of myometrial electrical activity was established. This method was used to study EMG activity in the uterus during the various stages of the oestrous cycle in mares. Recordings were analysed from four pairs of electrodes that were surgically implanted in the myometrium of four reproductively sound mares. The electrodes were placed at the tip, middle and base of the left horn and in the uterine body. Electrical activity was monitored by a polygraph. Data were transformed to a digitized form and statistically analysed. Myometrial electrical activity during oestrus and dioestrus was analysed for frequency (number of activity bursts per hour), duration (time elapsed from the start to the end of a burst) and intensity (peaks per minute). In addition, the degrees of synchronous activity among all of the monitored sites of the uterus were compared. The minimal amount of time of EMG recordings that allowed meaningful statistical analyses was determined to be 3.5 h during oestrus and 7 h during dioestrus. Variations in intensity of electrical activity were seen between the sites of implanted electrodes (P < 0.001). Electrical activity was highest in the middle of the horn and lowest in the uterine body. The frequency of activity bursts was the same during oestrus and dioestrus (4.35 ± 0.22 and 4.44 ± 0.20, respectively). The duration and intensity of uterine electrical activity depended on the stage of the oestrous cycle. The mean duration of uterine activity bursts during dioestrus was significantly (P < 0.005) shorter during oestrus (3.32 ± 0.18 min) than during dioestrus (5.7 ± 0.39 min). The intensity of bursts was higher during oestrus than during dioestrus (P < 0.005). In contrast, the total time of uterine activity was higher during dioestrus than during oestrus (P < 0.005). Synchronization of uterine electrical activity among different sites of the uterus was more marked during oestrus than during dioestrus (P < 0.005). It was suggested that a hormone-dependent mechanism for the regulation of cell communication is responsible for at least part of the changes of myometrial electrical activity that was observed at different stages of the oestrous cycle. It was concluded from the study that the method described for analysing EMG data allowed for an objective and repeatable interpretation of myometrial electrical activity.

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J. F. Kirkpatrick, I. M. K. Liu, J. W. Turner Jr, R. Naugle and R. Keiper

Summary. Ten feral mares free-roaming in Maryland, USA, were inoculated with porcine zonae pellucidae (PZP) protein before the breeding season for three consecutive years (1988–90). Ovarian function was monitored for 51 days during the peak of the breeding season after the third annual PZP inoculation, in seven of these mares and in four untreated control mares, by means of urinary oestrone conjugates and nonspecific progesterone metabolites. None of the ten inoculated mares became pregnant in 1990, compared with 55% of 20 control mares, which included two of the four monitored for ovarian function. Three of the untreated mares demonstrated apparent normal ovarian activity, characterized by preovulatory oestrogen peaks, concurrent progesterone nadirs at ovulation, breeding activity, and luteal-phase progesterone increases after ovulation. Two of the seven monitored PZP-treated mares demonstrated ovulatory cycles that did not result in conception. One was pregnant as a result of conception in 1989 and demonstrated a normal, late-gestation, endocrine profile. The remaining four PZP-treated mares revealed no evidence of ovulation, and urinary oestrogen concentrations were significantly depressed. The experiments indicated that (i) a third consecutive annual PZP booster inoculation is > 90% effective in preventing pregnancies in mares and (ii) three consecutive years of PZP treatment may interfere with normal ovarian function as shown by markedly depressed oestrogen secretion.

Keywords: zona pellucida; contraception; ovary; immunocontraception; oestrogen; progestin; horse

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C. G. Gravance, I. K. M. Liu, R. O. Davis, J. P. Hughes and P. J. Casey

The heads of stallion spermatozoa were analysed by computer automated sperm head morphometry and the morphometric values of the major subpopulations of sperm heads were assessed. The criteria for normal dimensions of stallion sperm heads are proposed based on the analysis of these measurements. Semen samples were collected from 10 fertile and 10 subfertile stallions, processed by a standard method, smeared onto microscope slides and stained using haematoxylin. At least 200 properly digitized sperm heads were analysed from each stallion. The measurements for length, width, area, perimeter and width/length were recorded for each stallion. All sperm head measurements were placed in a statistical database and multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters of fertile and subfertile stallions were compared by analysis of variance. The ranges of the values of the major clusters of fertile stallions were applied to all stallions to determine the percentage of normal sperm heads for each stallion. The mean values for length, width, area and perimeter in the major cluster of sperm head dimensions of fertile stallions were significantly different from those of the subfertile stallions (P < 0.001). The range of values of the major cluster of fertile stallions was length = 4.9–5.7 μm, width = 2.5–3.0 μm, width/length = 0.45–0.59, area = 10.3–12.1 μm, and perimeter = 12.9–14.2 μm. On the basis of these values, a significantly (P < 0.001) higher percentage of normal sperm heads were found in the fertile group than in the subfertile group of stallions (52% versus 19%).

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J. W. Turner Jr, I. K. M. Liu and J. F. Kirkpatrick

Regulation of local overpopulations of free-roaming feral equids is in demand worldwide for ecological balance and habitat preservation. Contraceptive vaccines have proven effective in feral horses, which breed seasonally, but no data are available for equids such as the burro, which is reproductively active all year round. In the present study, 27 individually identified female feral burros (Equus asinus) roaming free in Virgin Islands National Park (St John, US Virgin Islands; Lesser Antilles) were remotely treated with pig zonae pellucidae (PZP) vaccine. Between January and May, 16 burros were darted with a 1 ml emulsion of PZP plus Freund's adjuvant. Ten to twelve months later each treated burro was given a single booster injection of PZP plus adjuvant to maintain contraception through a second year. Eleven adult untreated jennies served as controls. Beginning one year after initial vaccination, these burros were monitored for pregnancy and foal production. Collection of data to determine treatment effect was not begun until 12 months after initial treatment to ensure that pregnancies existing before vaccination were not included. Pregnancy was assessed using previously validated methods for steroid metabolite measurement in fresh faecal samples. None of the PZP-treated burros produced foals between 0 and 12 months after the last inoculation. One PZP-treated burro tested positive for pregnancy at 10 months after the final inoculation. During this same period, six of 11 untreated burros tested pregnancy-positive, and four were observed with foals. There was no difference in pregnancy rates among treated, control and randomly sampled jennies between 12 and 24 months after the last inoculation. The results demonstrate that, in free-roaming feral burros that are reproductively active all year round: (1) burros can be accessed for remotely delivered PZP vaccination; (2) PZP contraception is effective; (3) PZP contraception is reversible; and (4) pregnancy can be reliably detected by faecal steroid analysis.

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I K M Liu, JW Turner Jr, E M G Van Leeuwen, D R Flanagan, J L Hedrick, K Murata, V M Lane and M P Morales-Levy

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I K M Liu, J W Turner Jr, E M G Van Leeuwen, D R Flanagan, J L Hedrick, K Murata, V M Lane and M P Morales-Levy

In this study of equids, we investigated the antibody response and the effect on the estrous cycle following a single inoculation of porcine zonae pellucidae (pZP) employing controlled-release methodology. We also investigated the use of two different water-soluble adjuvants as an alternative to oil-based adjuvants. Twenty-seven domestic mares were inoculated with various formulations of pZP and adjuvant. We showed that the anti-pZP antibodies generated as a result of the inoculations persisted for at least 43 weeks (length of the study). Of the various formulations used in the study, pZP and QS-21 water-soluble adjuvant, administered in combination with an emulsified preparation of pZP and Freund’s Complete Adjuvant generated a significantly (P < 0.05) higher titer of anti-pZP antibodies when compared with other formulations employing the water-soluble adjuvant, Carbopol. Hormone analyses for cyclicity indicated a high incidence and extended duration of persistent corpora lutea among the treated mares. The positive control group of mares receiving two standard inoculations of pZP and Freund’s Complete and Incomplete Adjuvants, as well as the placebo group of mares injected with QS-21 only, also exhibited high incidences of persistent corpora lutea. However, all mares eventually returned to normal cyclicity. The basis for the high incidence and extended duration of persistent corpora lutea was unexplained. The results demonstrate for the first time the persistent generation of anti-pZP antibodies following a single inoculation of pZP incorporated into a controlled-released preparation in the horse. This study further suggests that a single inoculation of pZP sequestered in a controlled-release lactide-glycolide polymer may serve as an alternative to traditional two-inoculation protocols for contraception investigations in the equine.