Cryopreservation of Embryos of Farm Animals

This review contains two parts. The first part is devoted to the significant steps in cryopreservation of mammalian embryos with emphasis on cattle and sheep that serve as models of reference. These steps are: (1) shortening of cooling and warming processes; (2) addition and dilution of cryoprotectant in one step; (3) introduction of plastic straw as a freezing and dilution container; (4) the choice of ethylene glycol as the quite universal cryoprotectant because of its low toxicity and high permeability; (5) vitrification, a cryopreservation method which enable passage from the liquid to the solid state by extreme elevation of viscosity due to high concentration of cryoprotectants and very rapid cooling. There are several vitrification solutions which contain dimethyl sulphoxide, glycerol, ethylene glycol, or a mixture of them, as basic cryoprotectants. The second part considers some factors affecting the efficiency of cryopreservation concerning (i) the origin of embryos and (ii) the stage of development and species. The origin of embryos (in vivo versus in vitro): in vitro embryos show a chilling and freezing sensitivity associated with their lipid content which can be modified by the culture conditions. Both conventional freezing and vitrification have been used and it seems that vitrification is more adapted to in vitro embryos when some modifications of initial protocols are carried out, particularly the rate of cooling. Thus considerable progress has been achieved by using the open pulled straw method of Vajta which enables the use of a minimum volume of freezing medium (0.5 μl) and a very high cooling rate that permits rapid traversal of the damaging temperature zone, corresponding to chilling sensitivity. The stage of development and species: not only are there differences between species at the same stage of development but in the same species all stages of development do not survive equally under the same freezing protocol. In cattle for example, oocytes and early stages of development in vivo or in vitro do not survive whereas compacted morulae and blastocysts survive very well. In the pig hatched blastocysts survive better than the other stages. Horse embryos have special characteristics that pose problems for successful freezing. In conclusion, a lot of work remains to be done to define fundamental characteristics of embryos of certain species (pig, horse) and of embryos of some stages or of oocytes.     

Direct and Residual Contributions of Symbiotic Nitrogen Fixation by Legumes to the Yield and Nitrogen Uptake of Maize (Zea mays L.) in the Nigerian Savannah

Field experiments were conducted to determine the direct and residual contributions of legumes to the yield and nitrogen (N) uptake of maize during the wet seasons of 1994 and 1995 at the University Farm, Abubakar Tafawa Balewa University, Bauchi, Nigeria, located in the Northern Guinea savannah of Nigeria. Nodulating soybean, lablab, green gram and black gram contributed to the yield and N uptake of maize either intercropped with the legumes or grown after legumes as a sole crop. Direct transfer of N from the nodulating soybean, lablab, green gram and black gram to the intercropped maize was 24.9–28.1, 23.8–29.2, 19.7–22.1 and 18.4–18.6 kg N ha^–1, respectively. However, the transfer of residual N from these legumes to the succeeding maize crop was 18.4–20.0, 19.5–29.9, 12.0–13.7 and 9.3–10.3 kg N ha^–1, respectively. Four years of continuous lablab cropping resulted in yields and N uptake of the succeeding maize crop grown without fertilizer N that were comparable to the yields and N uptake of the succeeding maize crop supplied with 40–45 kg N ha^–1 and grown after 4 years of continuous sorghum cropping. It may therefore be concluded that nodulating soybean, lablab, green gram and black gram may be either intercropped or grown in rotation with cereals in order to economize the use of fertilizer N for maize production in the Nigerian savannah.     

The Morphology of 16 and 17 Day Old Bovine Blastocysts from Virgin and Repeat Breeder Heifers

The morphology of 16–17 days old embryos from virgin heifers (VH) and repeat breeder heifers (RBH) was compared using light and electron microscopy. In addition some embryos transferred from one heifer category to the other were studied. Embryos from VH were elongated blastocysts and the oval embryonic disc had three germ layers. The ectoderm was stratified and many mitoses were seen. The endoderm lining the blastocoelic cavity consisted of almost squamous cells conjoined by tight junctions. Between the ectoderm and the endoderm the mesoderm had developed and expanded laterally and the coelom had formed. The trophoblastic cells adjacent do the embryonic disc were cylindrical, whereas those more peripheral located were cuboidal. The trophoblastic cells were conjoined by tight junctions and they had numerous long microvilli on their peripheral surface. Except in the embryonic disc region, the endodermal cells had filopodial processes towards the trophoblast. The embryos from RBH varied in appearance. One was similar to those from VH whereas the others were, more or less retarded, without formation of mesoderm. The smaller one consisted ot trophoblastic cells only. The transferred embryos (representing surviving embryos: 2 out of 9 in VH-RBH and 5 of 6 in RBH-VH) had a morphology similar to that of VH blastocytes two though, appeared somewhat retarded. It is suggested that the retarded embryos lack the ability to complete embryonic development and that the uterine environment of RBH is not favourable to sustain normal embryonic development.     

The immunohistochemical detection of Mycoplasma bovis and bovine viral diarrhea virus in tissues of feedlot cattle with chronic, unresponsive respiratory disease and/or arthritis.

The purpose of this study was to determine the frequency of selected pathogens in the tissues of a group of feedlot cattle with chronic disease (most often respiratory disease and/or arthritis). Samples of lung and joint tissues from 49 feedlot animals that had failed to respond to antibiotic therapy were tested by immunohistochemical staining for the antigens of Mycoplasma bovis, Haemophilus somnus, Pasteurella (Mannheimia) hemolytica, and bovine viral diarrhea virus (BVDV). Mycoplasma bovis was demonstrated in over 80% of cases, including in 45% of joints and 71% of lungs tested. Mycoplasma bovis was the only bacterial pathogen identified in the joints. Haemophilus somnus and Pasteurella (Mannheimia) haemolytica were found in 14% and 23% of cases, respectively, and were confined to the lungs in all instances. Infection with BVDV was demonstrated in over 40% of cases. Mycoplasma bovis and BVDV were the most common pathogens persisting in the tissues of these animals that had failed to respond to antibiotic therapy.     

Research on abalone (Haliotis midae L.) cultivation in South Africa

Although abalone fisheries in South Africa have existed since 1949, cultivation started by successful spawning of captured specimens in 1981 to produce spat and juveniles. Twelve abalone farms, with an estimated investment of US$12 million, have since been established on the coast of South Africa, with a projected production of 500–800 tons. While Haliotis midae could reach a maximum size of about 200 mm shell length at an age of over 30 years in the wild, farm production is concentrating on an average size of 100 mm after 5 years. Growth rates of 0.08–4.5% body weight day ^?1 for abalone of 10–17 mm shell length have been found under stimulated farming conditions on formulated diets, while the corresponding feed conversion ratio (FCR) was 0.9–2.4. Growth rate, FCR and protein efficiency ratio decline at water temperatures above 20 °C. Anaesthetics, for example magnesium sulphate, are used to prevent injury during removal from tanks. Prostrate diatoms, such as Cocconeis sublittoralis (Hendey), Amphora proteoides (Hustedt) and Achnanthes brevipes (Agardh) are preferred after the larval stage (5–7 days, depending on temperature) are finished. South Africa abalone growers prefer formulated feeds in pellet form, which is convenience and cost favourable to farm operations and management. Although fish meal has been found to be the most suitable protein source for inclusion in formulated diets, plant proteins such as soy bean meal, cottonseed meal and sunflower meal presented good growth and apparent protein digestibility. The parasite sabellid polychaete, Terebrasabella heterouncinata, recently named by Australian and American researchers, is indigenous to a variety of South African intertidal molluscs and impedes growth by causing irritation beneath the mantle in abalone. Prevention to some degree is possible by high standards of hygiene and husbandry of abalone in the tanks, but new techniques to control large infestations now include ultrasound. In spite of a reputation for toughness, abalone meat frozen immediately after shucking is tender relative to red meat. The success of abalone farming in South Africa has been as a result of a high degree of cooperation between the private sector and government‐backed research institutions, and it is anticipated that this collaboration will continue.     

Physeal Fractures in the Horse II. Management and Outcome

Seventy physeal fractures in horses were initially managed by euthanasia (18), stall confinement (25), application of a cast (7), or internal fixation (20). Of the 52 physeal fractures initially managed with stall confinement, a cast, or internal fixation, 23 (44%) healed and 13 (25%) of these horses became sound. The number of horses less than 4.5 months of age with pressure physeal fractures that became sound was significantly higher (p < 0.05) than the number of horses greater than 4.5 months of age. The number of horses with functional, pain-free limbs (sound horses) or functional limbs (lame horses) was not significantly different (p > 0.05) for Salter-Harris Type I, II, III, or IV pressure physeal fractures; however, critical examination for growth disturbances was not performed. The number of horses with pressure or traction physeal fractures of less than 5 days duration prior to presentation that healed or became sound was not significantly different (p > 0.05) when compared with those horses with fractures of greater than or equal to 5 days duration.     

An evaluation of the Olsen test as a measure of plant-available phosphorus in grassland soils derived from basalt parent material

Abstract. Anecdotal and circumstantial evidence have suggested that the Olsen test underestimates plant-available phosphorus (P) in basaltic soils in Northern Ireland. Therefore, the ability of this test to predict plant-available P in basaltic (and non-basaltic) soils was investigated by regressing Olsen-P data against herbage P indices calculated from plant tissue test data using the diagnosis and recommendation integrated system. The average Olsen-P concentration for a range of fields situated on basaltic soils was considerably lower than the average Olsen-P concentration for a range of fields situated on non-basaltic soils, and yet mean sward P status, as given by the herbage P indices, was similar for both groups of fields. Herbage P indices were also much better correlated with Olsen-P measurements in non-basaltic soils than in basaltic soils. Furthermore, at low Olsen-P values (≶9mgPL⁻¹) some swards on basaltic soils were genuinely deficient in P, while others were sufficient or even in surplus for this nutrient. The results confirm that Olsen-P is inadequate as a predictor of plant-available P in basaltic soils. It is concluded that an alternative soil test is needed to provide a reliable assessment of plant-available P in basaltic soils, to prevent overuse of fertilizer and manure P and to minimize the amounts of P entering local watercourses.     

Identification of the porcine cytomegalovirus major capsid protein gene.

A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified. Sequence analysis indicated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues. The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts. Phylogenetic analysis using herpesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse cytomegaloviruses. The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection. Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection. The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving virus-isolation are not useful.     

Doxorubicin affects the cardiac muscarinic system in the rat.

During the study on the mechanism of doxorubicin-induced cardiotoxicity, we observed that a long incubation (4 hr) with doxorubicin reduced the maximal negative inotropic effects of a muscarinic receptor agonist, carbachol. The mechanism responsible for this doxorubicin-induced reduction of the efficacy of carbachol was examined in isolated guinea pig hearts. In isolated left atrial muscle preparations, 1 hr incubation with 100 microM doxorubicin caused a parallel right-ward shift of the concentration-response curves for carbachol, but a longer (4 hr) incubation with this agent (30, 100 or 200 microM), caused a significant reduction of the magnitude of the negative inotropic effect of carbachol in addition to the concentration-dependent parallel right-ward shift. The 4-hr incubation with these concentrations of doxorubicin also reduced the maximal negative inotropic effect of an adenosine A1 receptor agonist, R-phenylisopropyl adenosine (R-PIA), without affecting the potency of this agonist. Doxorubicin (1 to 100 microM) reduced [3H]quinuclidinyl benzilate (QNB) binding in a concentration dependent manner, but failed to alter [3HIR-PIA binding. The decrease in the magnitude of the maximal negative inotropic effect by doxorubicin was caused by changes in the muscarinic system at steps common to the transduction of muscarinic and adenosine A1 receptor mechanisms.     

THE EFFECT OF A COMBINATION OF MEDETOMIDINE-BUTORPHANOL AND MEDETOMIDINE, BUTORPHANOL, ATROPINE ON GLOMERULAR FILTRATION RATE IN DOGS

The effects of intramuscularly administered medetomidine and butorphanol (MB), and medetomidine, butorphanol, atropine (MBA) on glomerular filtration rate (GFR) were determined in six dogs as measured by 99m-Tc-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) nuclear scintigraphy. Direct systolic, diastolic, and mean arterial blood pressures and heart rate were measured at regular time intervals before, during, and after GFR calculations. The mean GFR measurement following MB was significantly greater (4.44 ml/min/kg) than following MBA (3.82 ml/min/kg) or saline treatment (3.41 ml/min/kg). There was no significant difference between the mean GFR measurements following MBA injection and following saline injection. Diastolic and mean arterial pressures following MBA injection were significantly higher than the values recorded after either MB or saline alone. Heart rate following MB administration was significantly lower than that recorded for dogs receiving MBA or saline alone. The results of this study indicate that the administration of medetomidine in combination with butorphanol significantly increases total GFR in healthy dogs, while the administration of the combination of medetomidine, butorphanol, and atropine does not.