Serial investigations of early pregnancy in pony mares using real time ultrasound scanning

The uteri of 13 pony mares were examined daily by ultrasound during the first two months of gestation. The conceptus was first identified between 12 and 16 days after ovulation and the embryo was seen on the ventral surface of the conceptus after Day 21. The foetal heart could be visualised after Day 22 and spontaneous movement of the foetus occurred after Day 39. No consistent pattern was seen in the development of the foetal membranes, although attachment of the umbilical cord to the allantochorion was always on the dorsal aspect of the conceptus. Daily measurements were made of the diameter of the conceptus and the length of the embryo and foetus.     

Pregnancy diagnosis in cows with linear-array real-time ultrasound scanning: a preliminary note

Pregnancy diagnoses were performed under farm conditions in 201 cows (Hungarian Red Pied and crossbreds with Holstein) between 21 and 70 days after last insemination date, using a linear-array real-time ultrasound scanner with a 3.0 MHz rectal transducer. Identification of (a) sharply demarcated black area(s) and/or an embryo within the uterus were used as criterions for pregnancy. Pregnancy was confirmed by rectal examination at 6 and/or 8 weeks after A.I. From the total number of correct positive diagnoses (n = 129), incorrect positive diagnoses (n = 3, made on days 36, 40 and 44 after A I), correct negative diagnoses (n = 62) and incorrect negative diagnoses (n = 7, made on days 25, 28, 28, 29, 30, 31, and 33 after A I), a sensitivity of 94.8%, a specificity of 95,3%, a positive predictive value of 97.7% and a negative predictive value of 89.8% were calculated. From these preliminary results it was concluded that real-time ultrasound scanning is a useful and reliable technique for early pregnancy diagnosis in cows. It is suggested that a combination of milk/plasma progesterone estimation between days 21 and 24 and real-time ultrasound scanning between days 25 and 45 would allow the confirmation and quantification of (late) embryonic mortality in the cow.     

Plasma concentrations of 15-ketodihydro-PGF(2alpha), cortisol and progesterone during manual twin reduction in thoroughbred mares

The aim of the present study was to highlight the effect of two different techniques of one embryo crushing on some hormonal changes. Ten twinning mares were submitted to the mobile or fixed manual crushing of one blastocyst within day 19 after the last mating. Blood sample was collected from 20 min before to 90 min, 24 and 72 h after the procedure was performed to analyse 15-ketodihydro-PGF(2alpha), cortisol and progesterone plasma concentrations. Singleton pregnancy diagnosis was checked 72 h after crushing and at term of pregnancy. Because the unwanted crushing of both embryos occurred in one mare during the attempt of manual separation of the twins, that mare was not included in the evaluation of crushing-induced hormonal changes. No significant differences in hormonal concentrations were observed after one embryo crushing and also when the effect of the mobile (n = 6) or fixed (n = 3) technique was specifically evaluated. When the effect of the two techniques on each post-crushing sampling time hormonal levels was analysed, only a higher cortisol level 30 min after the fixed compared with the mobile technique was observed. The crushing performed within 19 days of gestation does not induce significant changes in 15-ketodihydro-PGF(2alpha), cortisol or progesterone plasma concentrations. When the fixed technique was performed, only a temporary higher cortisol concentration was seen 30 min after crushing, suggesting that the fixed technique might be responsible for a slight level of stress for the mare.     

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A ₄₅₀). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 ± 0.26 ng/ml. The cows in the ‘pregnant group’ had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.     

实时超声扫描对小型母马早期怀孕的连续检查的研究

应用Ａｌｏｋａ２１０Ｆ电子扫描超声诊断装置，频率为３．０ＭＨｚ的大家畜用直肠探头，对１３匹小型母马早期怀孕的胚胎发育进行了探查，最早在排卵后１２～１６ｄ探到了胎盘，２１ｄ可见胚胎，２２ｄ可见胎心搏动，３９ｄ可见胎动，虽然脐带与尿囊膜绒毛膜的联系总是在胎囊背侧，但胎膜的发育无固定形式，因此，应每天测定胎囊直径，胚胎及胎儿的长度。     

Comparative assessment of a double antibody enzyme immunoassay test kit and a triple antibody enzyme immunoassay for the diagnosis of Trichinella spiralis spiralis and Trichinella spiralis nativa infections in swine.

Enzyme immunoassays using the triple antibody enzyme linked immunosorbent assay (ELISA) with both Trichinella spiralis spiralis and T. spiralis nativa excretory-secretory (ES) antigens and a commercial Trichinella spiralis enzyme immunoassay test kit were carried out on sera from pigs that were infected with light, moderate and high doses of infective T. spiralis spiralis and T. spiralis nativa respectively. Seroconversion occurred in all pigs given infective Trichinella larvae although no trichinae were recovered from pigs given T. spiralis nativa larvae and examined between days 92 and 99 postinfection by pepsin digestion. Anti-Trichinella antibodies were detected in pigs infected with T. spiralis spiralis and T. spiralis nativa by ELISA using either the homologous or heterologous ES antigen. The commercial Trichinella spiralis enzyme immunoassay test kit also detected anti-Trichinella antibodies in both the T. spiralis spiralis and T. spiralis nativa infected pigs. The commercial test kit did not appear to be as sensitive as the triple antibody ELISA since it usually took two to three days longer for seroconversion to be detected by the former procedure. Finally seroconversion occurred more rapidly in swine infected with T. spiralis spiralis than with pigs receiving comparable doses of T. spiralis nativa.     

Pregnancy diagnosis in pigs: a field study comparing linear-array real-time ultrasound scanning and amplitude depth analysis

Between days 24 and 32 after mating/insemination, 881 pigs (785 pregnant and 96 not-pregnant) were tested for pregnancy on a commercial farm with a linear-array real-time ultrasound scanner. 5-7 Days later, 785 of these animals (708 pregnant and 77 not-pregnant) were tested again with A-mode equipment by farm employees. Confirmation of pregnancy was based on recorded farrowings or abortions; confirmation of non-pregnancy was based on return to oestrus and rebreeding, recorded non-farrowing, or inspection of the uterus of culled animals at the slaughterhouse. From the number of correct positive (a), incorrect positive (b), correct negative (c) and incorrect negative (d) diagnoses, a sensitivity (a/a + d) of 100% versus 97.5%, a specificity (c/c + b) of 90.6 versus 55.8%, a positive predictive value (a:a + b) of 98.9% versus 95.3% and a negative predictive value (c:c + d) of 100% versus 70.5% were calculated for the real-time ultrasound technique versus A-mode technique. It was concluded that real-time ultrasound scanning provides a very accurate technique for pregnancy diagnosis in pigs, enabling immediate decision making on treatment or culling of animals diagnosed as non-pregnant.     

Effect of thyrotropin releasing hormone (TRH) on serum levels of thyroid hormones in thoroughbred mares

Effects of thyrotropin releasing hormone (TRH) on serum levels of thyroid hormones were studied in 12 Thoroughbred mares. Significant increases (P<0.05) of serum T4 levels occurred as early as 2 hours and peaked at 4–10 hours after intravenous injection of 0.5 – 5 mg TRH. Following injection of 0.5, 1, 3 and 5 mg of TRH the serum levels of T4 were increased 2.25, 2.42,2.42 and 3.67 fold, respectively, over pre-injection levels. Serum levels of T3 were also significantly increased (P<0.05) at 1 or 2 hours and peaked at 2 to 4 hoursafter injection. The mean peak increase of T₃ levels were 2.87, 3.21, 3.10 and 3.10 fold over pre- injected in level in 0.5, 1, 3,5 mg treated horses, respectively. These results suggest that TRH can be an alternative to heterologous TSH for the equine thyroid function test. The recommended dosage is 1–3 mg, and most appropriate time to collect post-TRH blood sample is between 4–6 hours. Serum levels of T₄ and T₃ should increase 2–3 fold from baseline in normal horses.     

Pregnancy diagnosis in the dog: a comparison between abdominal palpation and linear-array real-time echography

This paper presents quantitative results of canine pregnancy diagnoses performed by abdominal palpation and/or linear-array real-time echography. Each animal was first palpated by an experienced clinician and then another investigator performed ultrasound scanning using a 3 MHz transducer. In other dogs echography was undertaken only when the result of abdominal palpation was negative or doubtful or when the owner requested ultrasound scanning. Using all tests performed between days 20 and 49 of pregnancy (day 0 = day of first mating or insemination), the results of abdominal palpation (n = 116) versus real-time scanning (n = 135) were: sensitivity: 89.0% v 92.9%; specificity: 93% v 96.0%; positive predictive value: 95.6% v 97.5%; negative predictive value: 83.3% v 88.9%. Results of ultrasound tests performed in 97 other dogs were: sensitivity: 92.4%; specificity: 97.7%; positive predictive value: 98.0% and negative predictive value: 91.4%. Almost identical figures were found when only the tests performed between days 25 and 35 of pregnancy were included. It was concluded that real-time ultrasound scanning is an accurate method for discriminating between pregnant and non-pregnant bitches; sensitivity and specificity of the abdominal palpation technique executed by an experienced clinician approach that of ultrasound scanning; small litter size and lack of knowledge of conception date are the main sources of false negative diagnoses.     

Rapid diagnosis of tuberculosis in dromedary camel (Camelus dromedarius) using lateral flow assay-based kit

Accurate early antemortem diagnosis of tuberculosis in dromedary camels is difficult due to the lack of reliable diagnostic test. The present study aimed to evaluate a lateral flow assay-based kit (rapid assay kit) in tuberculosis diagnosis that employs immuno-chromatographic detection of antibodies in serum, plasma, or whole blood. In a dromedary camel herd comprising 337 animals located at Bikaner, Rajasthan, India, 50 adult weak camels (11 males and 39 females) were tested by applying a single intradermal tuberculin test (SIDT) and rapid assay kit. A total of 14 animals (2 males, 12 females) were found positive in rapid assay. In SIDT, four animals revealed a positive reaction in the neck region and seven animals in the tail base. Another male animal was found SIDT positive but negative in rapid assay; it died after 12 months. Nine rapid assay positive animals died asymptomatically in 1- to 11-month period revealing postmortem tuberculosis lesions that were confirmed by Ziehl-Neelsen staining and histopathology. No tuberculous lesion was evident in the animal found positive in SIDT alone. Results of the present study indicated that serological tests like rapid assay kit can serve as a reliable test for antemortem diagnosis of tuberculosis in dromedary camel.

     

Accuracy of pregnancy diagnosis and prediction of calving date in red deer using real-time ultrasound scanning

One hundred and sixty-two 18-month-old farmed red deer were used to test the accuracy of pregnancy detection and equations for predicting gestational age. Deer ranging from 30 to 110 days gestation were examined by rectal ultrasonography using a 5 MHz transducer while they were standing. Each scan was recorded on video tape for measurements of uterine diameter, amniotic sac diameters, crown-rump length, head length, head diameter, nose length, chest depth, chest width and placentome base-apex length and width. Fetal age was calculated from the mean of the age predictions derived from each dimension measured on individual deer, for 132 deer between 44 and 110 days gestation. All the hinds diagnosed as pregnant produced offspring, and all the hinds diagnosed as not pregnant failed to produce offspring. Between one and six fetal and uterine dimensions were measurable and the number measurable increased with fetal age. The mean error of calving date prediction in 132 deer was 0.97 days. The error of prediction when measurements were made between 44 and 60 days was 0.44 days, whereas between 61 and 80 days and between 81 and 110 days the errors were +0.95 and +4.72 days, respectively. The estimates of calving date were all within 13 days of the calving date.     

Pregnancy and twinning rates in Thoroughbred mares following the administration of human chorionic gonadotropin (hCG)

AIM: To determine the effect of hCG administration to cycling Thoroughbred mares, on pregnancy and twinning rates and the number of serves in the treated cycle. METHODS: A retrospective case control approach was conducted involving 2119 mare ovulatory cycles, on 1110 mares over a 7-year period. Data were collected by 1 of the authors during routine stud work at 3 commercial Thoroughbred farms in the Waikato region of New Zealand. The hCG (1500 IU) was administered by intravenous injection to selected mares 24 h before the expected time of breeding. Mares were scanned for pregnancy (singleton or twins) 14 days after the onset of dioestrus or detection of ovulation. Multilevel logistic regression analyses were used to identify the risk factors associated with the outcomes of interest while simultaneously controlling for possible confounding factors. RESULTS: Treatment with hCG tended to improve the odds of pregnancy (p=0.06), produced a 3-fold increase in the odds of twins (p<0.001), and increased the odds of a mare having a single serve in the treated ovulatory cycle (p=0.036). The first ovulatory cycle of a season in which a mare was bred was associated with a lower odds of pregnancy (p=0.02), and a lower odds of twins (p=0.003), when compared with subsequent cycles. Lactating mares were less likely to be diagnosed with twins (p=0.005), and were more likely to have a single serve (p<0.001), in any one ovulatory cycle than non-lactating mares. CONCLUSIONS: This report supports the role of hCG as an important therapeutic tool in veterinary management of broodmares for optimal reproductive performance. Mares treated with hCG must be managed in the knowledge that they have an increased likelihood of twins.     

The detection of Bacillus anthracis protective antigens by enzyme immunoassay (EIA) using polyclonal and monoclonal antibodies

An Enzyme-Immunoassay (EIA) for the detection of Bacillus anthracis-protective antigen (PA) within one hour was developed. If the rabbit antiserum was used, 15 ng PA/ml could be detected and with the monoclonal antibody, the detection limit was 60 ng PA/ml. With respect to the higher specificity and with regard to the aspects of animalcare, monoclonal antibodies should be used in the test instead of the polyclonal antiserum.     

Superovulation in cycling mares using equine follicle stimulating hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation.In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation.Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares.In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3).In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Introduction

Induction of multiple ovulations or superovulation has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates.[1 and 2] Superovulation also has been suggested as a critical requirement for other types of assisted reproductive technology in the horse, including oocyte transfer and gamete intrafallopian transfer. [2 and 3] Unfortunately, techniques used successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone and equine chorionic gonadotropin have little effect in the mare. [4 and 5]The most consistent therapy used to induce multiple ovulations in mares has been administration of purified equine pituitary gonadotropins. Equine pituitary extract (EPE) is a purified gonadotropin preparation containing approximately 6% to 10% LH and 2% to 4% FSH.[6] EPE has been used for many years to induce multiple ovulations in mares [7, 8 and 9] and increase the embryo recovery rate from embryo transfer donor mares. [10] Recently, a highly purified equine FSH product has become available commercially.The objectives of this study were to evaluate the efficacy of purified eFSH in inducing superovulation in cycling mares and to determine the relationship between ovulation rate and pregnancy rate or embryo collection rate in superovulated mares.

Materials and methods

Experiment 1

Forty-nine normally cycling mares, ranging in age from 3 to 12 years, were used in a study at Colorado State University. Group 1 (control) mares (n = 29) were examined daily when in estrus by transrectal ultrasonography. Mares were administered an implant containing 2.1 mg deslorelin (Ovuplant, Ft. Dodge Animal Health, Ft. Dodge, IA) subcutaneously in the vulva when a follicle 35 mm in diameter was detected. Mares were bred with frozen semen (800 million spermatozoa; minimum of 30% progressive motility) from 1 of 4 stallions 33 and 48 hours after deslorelin administration. The deslorelin implants were removed after detection of ovulation.[11] Pregnancy status was determined at 14 and 16 days after ovulation.Group 2 mares (n = 10) were administered 25 mg of eFSH (Bioniche Animal Health USA, Inc., Athens, GA) intramuscularly twice daily beginning 5 or 6 days after ovulation was detected. Mares received 250 g cloprostenol (Estrumate, Schering-Plough Animal Health, Omaha, NE) intramuscularly on the second day of eFSH treatment. Administration of eFSH continued until a majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Mares were subsequently bred with the same frozen semen used for control mares, and pregnancy examinations were performed as described above.Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting 5 or 6 days after ovulation and were administered 250 μg cloprostenol on the second day of treatment. Mares were randomly selected to receive either a deslorelin implant (n = 5) or 2500 IU of human chorionic gonadotropin (hCG) intravenously (n = 5) to induce ovulation when a majority of follicles reached a diameter of 35 mm. Mares were bred with frozen semen and examined for pregnancy as described above.

Experiment 2

Sixteen cycling light-horse mares were used during the physiologic breeding season in Brazil. Reproductive activity was monitored by transrectal palpation and ultrasonography every 3 days during diestrus and daily during estrus. On the first cycle, mares were administered 2500 IU hCG intravenously once a follicle 35 mm was detected. Mares were subsequently inseminated with pooled fresh semen from 2 stallions (1 billion motile sperm) daily until ovulation was detected. An embryo collection procedure was performed 7 days after ovulation. Mares were subsequently administered cloprostenol, and eFSH treatment was initiated. Mares received 12 mg eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time hCG was administered. Mares were inseminated and embryo collection attempts were performed as described previously.

Statistical analysis

In experiment 1, 1-way analysis of variance with F protected LSD was used to analyze quantitative data. Pregnancies per ovulation were analyzed by x² analysis. In experiment 2, number of large follicles, ovulation rate, and embryo recovery rate were compared by Student,'s t-test. Data are presented as the mean S.E.M. Differences were considered to be statistically significant at p < .05, unless otherwise indicated.

Results

In experiment 1, mares treated with 25 mg eFSH twice daily developed a greater number of follicles 35 mm in diameter (p = .001) and ovulated a greater number of follicles (p = .003) than control mares (Table 1). However, the number of pregnancies obtained per mare was not significantly different between the control group and the group receiving 25 mg eFSH (p = .9518). Mares treated with 12 mg eFSH and administered either hCG or deslorelin to induce ovulation also developed more follicles 35 mm (p = .0016 and .0003, respectively) than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles (p = .003) than control mares, whereas the number of ovulations for mares receiving eFSH followed by deslorelin was similar to that of control mares (p = .3463). Pregnancy rate for mares induced to ovulate with hCG was higher (p = .0119) than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of controls (p = .692). Pregnancy rate per ovulation was not significantly different between control mares (54.5%) and mares treated with eFSH followed by hCG (52.9%). The lowest pregnancy rate per ovulation was for mares stimulated with 25 mg eFSH and induced to ovulate with deslorelin. The mean number of days mares were treated with 25 mg or 12 mg of eFSH was 7.8 ± 0.4 and 7.5 ± 0.5 days, respectively. Overall, 80.0% of mares administered eFSH had multiple ovulations compared with 10.3% of control mares.