Use of in vitro fertilization for production of calves from involuntary cull cows.

PURPOSE: The main purpose of the study was to assess the feasibility of a combined system for in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos for production of calves from cows that have to be removed prematurely from production units. RESULTS: Eighteen cows that were to be culled from experimental dairy production units were ovariectomized. An average of 45.7 oocytes per cow was collected from the ovaries. After in vitro maturation and fertilization of the oocytes, an average of 40.8 presumptive zygotes was placed into in vitro culture, with an average of 16.1 cleaving by day 2 and an average of 5.7 developing to morulae/blastocysts by day 6 or 7. A greater mean quantity of oocytes was collected from cows that were ovariectomized between day 5 and day 13 of the estrous cycle than from those that were ovariectomized between day 0 and day 3 of the estrous cycle. Correspondingly larger mean numbers of cleaved zygotes and morulae/blastocysts were produced from the cows that were ovariectomized between day 5 and day 13 of the cycle. Transferable embryos were produced from 17 of the 18 cows. Eighteen embryos from six oocyte donor cows were transferred to recipients. Six of the eighteen recipients were confirmed to be pregnant after 40 days. Three of the pregnant recipients delivered live calves at term. Two others remain pregnant but have not reached term. The sixth recipient aborted at approximately 120 days of gestation. CONCLUSIONS: Results from the preliminary study indicate that this system can be used for production of calves from cull cows. Although transferable embryos were produced from all except one cow, there was a high degree of variability among cows in total number of oocytes recovered and embryos produced. More donors need to be evaluated to determine the effects of age, breed, reason for culling, and source of semen.

Ultrasonography of the canine prostate with histologic correlation.

The present study assessed the canine prostate gland using B-mode real-time ultrasonography and correlated these findings with corresponding histologic sections. The prostate glands of 10 dogs were suspended in a saline bath and were scanned with an ultrasound probe. The glands were fixed and histologic sections were made. The sonograms were visually compared with the histologic sections. Areas having high collagenous tissue content appeared hyperechoic on the ultrasound scans. This hyperechogenicity was seen in the diffusely hyperechoic immature gland, the hyperechoic periurethral stroma, and the hyperechoic butterfly-shaped area seen on transverse scans. The butterfly-shaped area was isoechoic, with an elongated oval area seen on transverse scans. Areas of glandular epithelial content appeared hypoechoic on ultrasound scans. No zones comparable to the human prostate were observed.

The effects of FSH-priming and dominant follicular regression on the superovulatory response of cattle.

Thirty superovulatory treatments were administered to 19 mixed-breed, nonlactating cows. In 10 superovulatory treatments, the cows were primed with follicle stimulating hormone (FSH) on the second and third day of the estrous cycle, and in another 10 superovulatory treatments, the cows received no priming dosage of FSH. Initiation of the superovulatory treatments in both groups was determined by ultrasonically monitoring for regression of the dominant anovulatory follicle. Still another 10 superovulatory treatments were begun on Day 10 without regard for regression of the dominant anovulatory follicle and without a priming dosage of FSH. The mean days for starting the superovulatory treatment in the FSH-primed cows, in the nonprimed cows and in the controls were 10.5, 11.9 and 10 days, respectively. All cows were treated with eight injections of FSH at 12-hour intervals in a declining dosage (36 mg total). Cows were bred naturally and embryos collected nonsurgically seven days later. There was no significant difference (P>0.05) between the total number of embryos or transferable embryos in the three treatment groups. In this study neither priming on Days 2 or 3 nor initiating the superovulatory treatment, based on the morphologic regression of the dominant anovulatory follicle, was an effective means for improving the superovulatory response in cattle.

Effect of surgical and chemical in ovo bursectomy on lymphocyte density scores and histological evaluations of primary and secondary lymphoid organs in hypertensive and hypotensive turkeys.

The effects of surgical and chemical in ovo bursectomy on the histology of primary and secondary lymphoid organs were studied in hypertensive and hypotensive lines of turkeys. The effects of bursectomy were measured by determining the presence of lymphatic nodules and the presence of lymphocytes in secondary lymphoid organs: spleen, cecal tonsil, Meckel's diverticulum, and Peyer's patches; as well as in the thymus and bursa of Fabricius. No treatment effect on splenic nodule formation occurred, but a significantly lower lymphocyte density score was present in the surgically bursectomized group. Both nodule formation and lymphocyte density scores for the cecal tonsil and Peyer's patches were significantly reduced in the surgically bursectomized and testosterone-treated groups as compared with the control and sham groups. The most consistent depression in lymphatic scores in both the surgically and chemically bursectomized groups occurred in the Peyer's patches followed by cecal tonsils and Meckel's diverticulum. As expected, there were no differences in thymocyte density scores. There was no consistent effect on lymphatic scores due to blood pressure selection; however, the hypertensive line showed significantly lower scores than the hypotensive line in the bursa, cecal tonsils, and Peyer's patches. No differences between the sexes were noted in any organ.

Effect of surgical and chemical in ovo bursectomy on hatchability, mortality rate, and antibody response in hypertensive and hypotensive lines of turkeys.

The effect of in ovo bursectomy on hatchability, mortality rate, and antibody response to keyhole limpet hemocyanin (KLH) was studied in hypertensive and hypotensive lines of turkeys. Experiments were conducted to assess the optimal time to perform surgical in ovo bursectomy in turkey embryos for the lowest mortality rate. Factors such as the ease of operation, hatchability, and least deaths were considered. Surgical bursectomies at 25, 24, 23, and 22 day embryonation resulted in 75, 20, 0, and 10% hatchability, respectively. The surgical procedure performed at 25-day embryonation resulted in superior hatchability and ease of operation, and it was less traumatic as evidenced by fewer deaths. The hatchability of embryos injected with testosterone (TE) at 15 days incubation was 55%. Birds subjected to surgery or chemical treatment had increased mortality rates. Cyclophosphamide (CY) treatment at 1 and 2 days posthatch caused a significant increase in deaths over groups not treated with CY. Both primary and secondary KLH responses were significantly lower in bursectomized and TE-injected groups, than in shamoperated or nonoperated control groups.

Preliminary implications of B-mode ultrasonography of the testicles of beef bulls with normal breeding soundness examinations.

Four polled Hereford bulls were found to have satisfactory breeding soundness examinations. They were examined by B-mode ultrasonography, at which time the testicular diameter was measured by ultrasound. The testicles were removed, measured physically and this data was compared with the ultrasound measurements and correlated with other parameters of the breeding soundness examination. The testicles from bulls with normal breeding soundness examinations appeared ultrasonographically identical with the normal testicles from other species such as the pig and dog. Results indicated that the bull testicle diameter could be accurately measured by ultrasonography. Neither ultrasonographic nor physical testicular diameter measurements correlated statistically with scrotal circumference, but they did correlated well with testicular circumference, weight and volume.

Monolayer culture of cells originating from a preimplantation bovine embryo.

The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin, and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm2 had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged. Expansion to a 25-cm2 flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1:2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes.

Ultrasonographic evaluation of normal boar testicles.

The testicles of 14 Landrace boars (7 at 9 months of age and 7 at 15 months of age) were measured and examined by routine physical examination. Semen analysis was done. The testicles were then examined and measured by B-mode ultrasonography. The ultrasonographic appearance and measurements were compared with the physical measurements and the semen analysis. Seminiferous epithelial area (SEA) data were obtained after the testicles were surgically removed. These data were compared with the ultrasonographic data and the other physical data. A correlation could not be found between semen analysis and either physical or ultrasonographic data or between SEA data and semen analysis. Significant differences were found between the 9-month-old and the 15-month-old boars relative to 5 measurements: SEA, tunica albuginea thickness, testicular diameter, testicular length, and epididymal diameter. A ratio of 4:1 was found between each of the average physical measurements (scrotal diameter, testicular-epididymal circumference, testicular-epididymal length) and ultrasonographic measurements of testicular diameter. A ratio of 2.6:1 was found between testicular diameter and epididymal tail diameter (cranial to caudal).

Effects of embryo-freezing and thawing techniques on the survivability of Brucella abortus.

A suspension of a pathogenic strain (2308) of Brucella abortus was aliquoted, centrifuged, resuspended in 6 treatment media and quantitated. Ten 1-ml samples of each treatment were subjected to a standard embryo-freezing technique. The treatments were selected to examine the effects of 1) freezing and thawing, 2) cryoprotectants and 3) antibiotics on the survivability of Brucella suspended in embryo-support media. Five samples of each treatment were thawed and quantitated after a 2-wk storage period and five samples were thawed and quantitated after a 6-mo storage period. Means and percent reductions were determined for each treatment. There was no statistical difference between means at 2 wk and 6 mo within any treatment. Freezing and thawing caused a 64% reduction in the number of viable Brucella . The addition of antibiotics caused a 99.9% reduction in viability of the organism. Glycerol protected the organism during freezing and thawing in the absence of antibiotics but did not interfere with the high percent reduction seen when antibiotics were present. Dimethylsulfoxide (DMSO), however, not only protected the organism during freezing and thawing but also appeared to negate the effects of the antibiotics.

Microscopic characteristics of genital end bulbs in the penis of bulls.

Light microscopic sections of Epon-embedded bull penis were examined for nerve endings. Tissue was taken from the dorsal midline of the glans penis and from the free portion of the penis in 6 mature bulls. Genital end bulbs were identified in both regions of the penis and were located from 1 to 500 micron beneath the epithelium, with most end bulbs concentrated next to the epithelium in the glans penis. Occasionally, end bulbs were found within nerve trunks. Other encapsulated endings were not seen. Ultrastructural correlation of the light and electron microscopic appearance of spherical end bulbs and elongated end bulbs indicated that their structure was identical. Two to 6 neurites filled with mitochondria were surrounded by several layers of thin cytoplasmic lamellae. The lamellae were characterized by pinocytotic vesicles, microtubules, and a scant basal lamina. A capsule of differing thickness enclosed the end bulbs. Fewer capsular layers surrounded more superficially located end bulbs. Near the epithelium, there were gaps in the capsule. Nonmyelinated fibers were seen within end bulbs and between end bulbs and the epithelium.

The effects of superovulation on Brucella abortus infection in the bovine uterus.

Uterine flushings were collected three times at predetermined intervals from 11 mixed-breed beef cows and cultured for Brucella abortus . Prior to sampling, all cows had aborted fetuses from which brucellae had been isolated. Initial collections were made between 21 and 34 days following abortion. The second flushing was conducted at the onset of injections used for inducing superovulation and the third flushing was conducted 6 to 8 days after the ensuing estrus. The latter two flushes were conducted between 60 and 120 days following abortion. Brucellae were isolated from uterine flushings collected from 6 of the 11 cows on the initial round of sampling. Cultures of all subsequent uterine flushings collected before and after injections for superovulation were negative. It was concluded that the superovulatory treatment is not likely to reactivate the release of brucellae into the uterine lumen during the period when embryos are normally collected.

Surgical technique to cannulate the rete testis of the goat.

A surgical technique was devised to collect rete testis fluid from 14 mature goats. The tubular portion of the extratesticular rete testis was cannulated where it penetrated the tunica albuginea of the testis under the center of the head of the epididymis. The tip of the cannula was sutured in place in the extratesticular rete testis and the opposite end was passed through the scrotum and into a plastic collection bottle. The bottle was glued to pillow ticking, which was sutured to the scrotum. Continuous flow of rete testis fluid occurred for 0 to 14 days and was usually terminated by a sperm clot in the cannula. The flow rate was 0.59 +/- 0.37 ml/hr.

Effect of deprival of rete testis fluid on the morphology of efferent ductules.

One week after unilateral cannulation of the rete testis and ligation of the efferent ductules, samples of the proximal, middle and distal segments of the efferent ductules of 6 goats were examined by light and electron microscopy and compared with normal contralateral efferent ductules. The pseudostratified columnar epithelium consisted of ciliated, nonciliated and basal cells. The number of clear vacuoles decreased markedly in the proximal and middle segments following deprivation of androgen-rich rete testis fluid. The epithelium of the distal segment of the cannulated side had few large clear vacuoles compared to the normal side, which had a high concentration of large vacuoles. Since the large vacuoles decreased in all three segments following ligation, they were thought to be absorptive. Some cells of the distal segment of the cannulated side contained a single, huge, basal vacuole. Electron-dense, membrane-bound granules were abundant in the proximal segment of normal ductules. After cannulation these granules were still present. It was concluded that the electron-dense granules were insensitive to rete testis fluid and that they did not arise from the fluid leaving the rete testis.

Culture of uterine flushings, cervical mucus, and udder secretions collected post-abortion from heifers artificially exposed to Brucella abortus.

Uterine flushings, cervical mucus swabs and udder secretions collected at weekly intervals from five mixed breed beef cows (four Brucella abortus strain 19 vaccinates, and 1 non-vaccinate) were cultured for Brucella abortus . Prior to sampling, four of the five had aborted 7-to 8-month-old fetuses and one gave brith to a weak calf. The fetuses and/or udder secretions from the cows were culture positive for B. abortus at the time of parturition. Three of the cows developed persistent udder infections. Two of these cows were also shown to have brucellae in their cervical mucus for 10 and 20 days and in their uterine flushings for 17 and 41 days after parturition, respectively. One other cow had brucellae in the cervical mucus for 16 days and in the uterine flushings for up to 36 days post-abortion. All attempts to isolate the organism from this cow's udder secretions in culture were negative. In two cows with culture-positive uterine flushings, isolations of brucellae were made subsequent to normal postpabortion return to estrus.

Clinical approach to determine the contribution of the palmar and palmar metacarpal nerves to the innervation of the equine fetlock joint.

To determine the sensory nerve supply of the metacarpophalangeal joint, lameness was induced in eight horses by injecting the joint with a glycerin suspension of glass micropheres. When the medial and lateral palmar nerves were anesthetized in 4 horses, there was noticeable improvement in the gait, but each horse remained lame. When the medial and lateral palmar metacarpal nerves were also anesthetized, 3 of the 4 horses became sound. To confirm the results of local anesthesia, neurectomies were performed on a second group of four horses. The lameness was alleviated only upon resection of both the palmar nerves and the palmar metacarpal nerves.

Studies on infectious bursal disease in chickens. 2. Scoring microscopic lesions in the bursa of fabricius, thymus, spleen, and kidney in gnotobiotic and battery reared White Leghorns experimentally infected with infectious bursal disease virus.

During a 2 1/2 year study on the effect of experimentally-induced infectious bursal disease (IBD) in White Leghorns, a system for scoring microscopic lesions was developed. Microscopic changes in the bursa of Fabricius, thymus, spleen, and kidney were studied to create a lesion score for each organ. No changes occurred in the cecal tonsil and few, if any, changes occurred in the liver. The system was useful in evaluating the severity of pathologic and inflammatory changes in each organ during the course of the disease. Lesion scores for the bursa ranged from 0 for normal bursae to +4 for severely affected bursae. The lesion scores for the thymus, spleen, and kidney ranged from 0 to +3. The criteria for scoring lesions in the thymus were: 0 equaled no change; +1- cortex had a few empty spaces, pronounced hyperemia with some heterophil infiltration, especially in the medulla; +2- cortex had an increase in the number of empty spaces and increased heterophil infiltration, and the cortex and medulla had decreased hyperemia; +3- cortex had numerous round aggregations (12 to 16 mu in diameter) of cell debris and pyknotic nuclei, a definite decrease in the lymphocyte density in the cortex, and decreased hyperemia in cortex and medulla. This lesion scoring system was useful in determining the severity of IBD in different-aged progeny from IBD-immune and nonimmune dams.

Histochemical localization of adenosine triphosphatase activity in bovine ruminal epithelium.

A fine-structural histochemical technique was used to localize magnesium-dependent adenosine triphosphatase (Mg-ATPase) activity in ruminal mucosa. Precipitate appeared on the cytoplasmic surface of the plasmalemma in cells of the upper stratum spinosum, the stratum granulosum, and the deepest layer of the stratum corneum. This ATPase activity was sensitive to glutaraldehyde fixation and possibly to ouabain, but was unaffected by sodium and potassium. The preponderance of Mg-ATPase activity in bovine ruminal epithelium may make it impossible to detect sodium-potassium-activated adenosine triphosphatase ((Na + K)-ATPase) activity histochemically. A Mg-ATPase activity also occurred in mitochondria of the stratum spinosum and stratum granulosum. None of the ruminal sections hydrolyzed adenosine diphosphate, inosine triphosphate, or beta-glycerophosphate when these compounds were used as substitute substrates for adenosine triphosphate. When adenosine-5'-monophosphate was the available substrate, a reaction product appeared in the same layers as Mg-ATPase activity, but the reaction product was confined to the intercellular space.