Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses

Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. Mechanisms controlling neurohypophysial hormone release in the rat

To explore mechanisms of osmotic control of neurohypophysial hormone release, electrical activity of magnocellular neurons (MCNs) in the hypothalamus, related neurons and efferent renal sympathetic nerve activity (RSNA) were recorded from urethane-anesthetized rats. Local osmotic stimulation applied to organum vasculosum of the lamina terminalis (OVLT) or median preoptic nucleus (MnPO) excited MCNs. Although OVLT neurons projected to MCNs were unresponsive to hyperosmotic stimulation, those projected to MnPO and also receiving excitatory inputs from MCNs, were excited by it. MnPO neurons, which were driven by OVLT stimulation and projected to MCNs, were also osmosensitive. Excitatory connections thus exist from MCNs to OVLT, from OVLT to MnPO and from MnPO to MCNs. Neurons in each of these connections were osmosensitive. This circuit thus appears to constitute an osmoreceptor complex essential for the osmoreception of MCNs. MnPO neurons constituting a part of the osmosensitive circuit were also sensitive to hemodynamic change. Thus this circuit may integrate hemodynamic and osmotic information. Local anesthesia of MnPO diminished activation of RSNA and pressor response induced by third cerebroventricular injection of hypertonic saline. The results suggest that the osmosensitive circuit is involved in body fluid regulation not only by controlling vasopressin secretion but also by modulating sympathetic outflow.     

Structural changes in intramuscular connective tissue during the fattening of Japanese black cattle: effect of marbling on beef tenderization

We investigated changes in structures and mechanical properties of the intramuscular connective tissue during the fattening of Japanese Black steers, using the cell maceration method for scanning electron microscopy. During the early fattening period, from 9 to 20 mo of age, collagen fibrils of the endomysium in longissimus muscle associated more closely with each other, and collagen fibers in the perimysium increased in thickness and their wavy pattern became more regular. These changes were closely related to the increase in mechanical strength of the intramuscular connective tissue and resulted in a toughening of the beef during the period. The shear force value of longissimus muscle decreased after 20 mo of age, concomitantly with the rapid increase in the crude fat content. Scanning electron micrographs of the longissimus muscle dissected from 32-mo-old steers clearly showed that the adipose tissues were formed between muscle fiber bundles, that the honeycomb structure of endomysia was partially broken, and that the perimysium separated into thinner collagen fibers. In semitendinosus muscle, in which the crude fat content was lower (P<.05) than that in longissimus muscle, the structure of the intramuscular connective tissue remained rigid at 32 mo of age. The shear force value of the muscle increased even in the late fattening period, from 20 to 32 mo of age. Thus, the development of adipose tissues in longissimus muscle appears to disorganize the structure of the intramuscular connective tissue and contributes to tenderization of highly marbled beef from Japanese Black cattle during the late fattening period.     

威宁县肉牛产业发展现状及对策

威宁县素有南方“畜牧之乡”的美誉,作为贵州肉牛产业发展的重点区域,威宁县按照“三年打基础、五年创品牌、十年磨一剑”的工作思路,扎实推进肉牛产业发展,取得了明显成效。但仍存在养殖方式粗放、产销融合不够、利益联结不充分、良种繁育体系不健全等问题。笔者结合实际,深入剖析现阶段威宁县肉牛产业发展现状及存在的问题,并提出有关对策,旨在为下一步威宁县肉牛产业高质量发展提供参考。     

The prevalence of antimicrobial susceptibility of veterinary pathogens isolated from cattle and pigs: national antibiotic resistance monitoring 2002/2003 of the BVL

The national antimicrobial resistance monitoring determines the current quantitative resistance level of life-stock pathogens, in order to permit the evaluation and surveillance of the distribution of resistances on a valid basis. During the examination period from June 2002 to July 2003, a total of 1849 pathogens was collected, following a representative German-wide pattern in collaboration with 29 laboratories. The selection of examined bacterial strains included different specimen causing respiratory diseases in fattening pigs (Pasteurella multocida, Bordetella bronchiseptica) and cattle (Pasteurella multocida, Mannheimia haemolytica), respectively, as well as strains causing mastitis in dairy cows (Staphylococcus spp., Streptococcus spp., E. coli). Determination of the in-vitro susceptibility (minimal inhibitory concentration) to at least 17 antimicrobial agents was performed centrally by the BVL using the microdilution broth method. The findings approximately match results of an analogous study conducted in 2001, and correspondingly revealed significantly lower resistance-values in comparison to data published for Germany so far. No correlation could be established between the incidence of resistance and differing stock densities. By means of the resistance monitoring data gathered by the BVL, the risk potential of antimicrobials applied in Germany can be reliably defined. This valuable information about the epidemiological situation of resistance in Germany can be helpful to veterinarians as a decision guidance when choosing appropriate means of therapy. Accordingly, the results stated by the BVL are apt to provide an important contribution to improving the safety of food-animal products. The experiences obtained from the monitoring also show that valid data about the antimicrobial susceptibility can only be raised on an interdisciplinary approach (federal agencies, county diagnostic laboratories, universities, industry). Currently, the BVL put into practice a study with an extended spectrum of bacterial species and indications.     

Decreasing the roughage:concentrate ratio of a diet to determine the critical roughage part for beef cattle.

The critical roughage part (CRP) of 2 diet types was determined in a cross-over design with 6 double-muscled and 6 normally conformed Belgian Blue bulls fitted with rumen cannulae. The roughage:concentrate ratio was lowered weekly until signs of a lack of physical structure were observed. For diet 1, consisting of maize silage and concentrates, the initial proportion of maize silage was 25% of DM but it decreased weekly with 5% units of DM. For the second diet, consisting of wheat straw and concentrate, 12% straw (DM basis) was provided during the first week and thereafter the proportion of straw decreased weekly with 3% units of DM. Several directly observable parameters (rumen pH, feed intake, bloat, faecal consistency) were evaluated weekly for each bull. Apart from these direct indicators of acidosis, also other parameters, whose results were only available after the end of the trial, were determined (volatile fatty acid profile, lactic acid concentration, chewing time). The roughage part between the part fed when signs of a lack of physical structure was first observed and the part that was fed the week before, was considered as the CRP. Most animals showed no acute signs of clinical acidosis (directly observable parameters) and finished the trial on a 100% concentrate diet. However, in sacco rumen DM-degradabilities of maize silage, grass silage and wheat grain was depressed considerably when low roughage diets were fed. Based on all observed parameters, the mean CRP was calculated to be 14.7% for diet 1 and 8.1% for diet 2. The beef type (double-muscled or not) had no influence on the CRP.     

A computer model to predict the effects of level of nutrition on composition of empty body gain in beef cattle: I. Theory and development.

A computer model was developed to simulate differences in the composition of empty body gain of beef cattle resulting from differences in postweaning level of nutrition that are not associated with empty BW. Differences in rate of empty body gain of cattle of similar genotype reflect differences in the level of nutrition; hence, empty body gain was used to represent level of nutrition. The model was based on four assumptions: 1) as animals mature, the proportion of fat is greater in gain than in body weight, 2) effects on body composition caused by plane of nutrition that are not associated with empty BW are predictable from rate of empty BW gain, 3) effects resulting from changes in nutrition are not exerted immediately nor are they permanent, and 4) cattle approach an empty body composition equilibrium when empty body gain is zero. Six parameters were used to characterize mature fat-free matter, rate of change to body composition equilibrium, minimum and maximum fractional growth rate relative to fractional growth rate for empty BW, time lag of response to change in nutrition, and influence of stage of maturity and nutrition on rate of change for fat-free matter. Two parameters were specific for genotype and gender. Using results from the model, we were able to explain conflicting results obtained from compensatory gain experiments.     

Effect of the polyvinylpyrrolidone concentration of cryoprotectant on mouse embryo development and production of pups: 7.5% of PVP is beneficial for in vitro and in vivo development of frozen-thawed mouse embryos

In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 mul of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO(2) in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.     

ASAS centennial paper: Contributions in the Journal of Animal Science to the development of protocols for breeding management of cattle through synchronization of estrus and ovulation

American Society of Animal Science members, publishing in Journal of Animal Science (JAS), completed research that resulted in understanding the estrous cycle of cattle, which led to the ability to inseminate cattle on a given day with pregnancy rates similar to those achieved by 21-d breeding by a fertile and sound bull. Research published in JAS led to understanding estrus, ovulation, the estrous cycle, and postpartum interval for cattle (1930s through 1960s) and hormonal factors affecting corpus luteum lifespan of cattle (1950s through 1980s). Research during the 1940s to 1960s, using gonadotropins and progesterone to manage the estrous cycle of cattle, established the concepts for estrous synchronization and stimulated commercial research directed at developing cost-effective progestogen estrous synchronization products, leading to commercially available products from 1967 through today (Repromix, melengestrol acetate, Syncro-Mate-B, controlled internal drug release). Prostaglandin F(2alpha) products were approved for estrous synchronization (1970s, 1980s), and GnRH products were approved for use in cattle to treat ovarian follicular cysts (1970s, 1980s). Research published in JAS was essential for understanding the biology of and potential value of both PGF(2alpha) and GnRH and contributed both to new knowledge and scientific bases for future Food and Drug Administration Center for Veterinary Medicine approval of those products. Research during the1980s through 2000s led to understanding ovarian follicular waves and described the timing of follicular recruitment, selection, dominance, and atresia; this research was essential for the ability to effectively manage follicles to achieve success with timed AI. The knowledge gained through research published in JAS resulted in development of the numerous estrous synchronization and breeding management protocols that are cost-effective and meet the breeding management needs of most beef and dairy enterprises.     

The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing.

Fecal samples collected from cattle at processing during a 1-year period were tested for verotoxins (VT1, VT2), Escherichia coli O157:H7, and Salmonella. Verotoxins were detected in 42.6% (95% CI, 39.8% to 45.4%), E. coli O157:H7 in 7.5% (95% CI, 6.1% to 9.1%), and Salmonella in 0.08% (95% CI, 0.004% to 0.5%) of the fecal samples. In yearling cattle, the median within-lot prevalence (percentage of positive samples within a lot) was 40% (range, 0% to 100%) for verotoxins and 0% for E. coli O157:H7 (range, 0% to 100%) and Salmonella (range, 0% to 17%). One or more fecal samples were positive for verotoxins in 80.4% (95% CI, 72.8% to 86.4%) of the lots of yearling cattle, whereas E. coli O157:H7 were detected in 33.6% (95% CI, 26.0% to 42.0%) of the lots. In cull cows, the median within-lot prevalence was 50% (range, 0% to 100%) for verotoxins and 0% (range, 0% to 100%) for E. coli O157:H7 and Salmonella (range, 0% to 0%). Verotoxins were detected in one or more fecal samples from 78.0% (95% CI, 70.4% to 84.2%) of the lots of cull cows, whereas E. coli O157:H7 were detected in only 6.0% (95% CI, 3.0% to 11.4%) of the lots of cull cows. The prevalence of verotoxins in fecal samples was lower in yearling cattle than in cull cows, whereas the prevalence of E. coli O157:H7 in fecal samples was higher in yearling cattle than in cull cows. The prevalence of E. coli O157:H7 in fecal samples was highest in the summer months. Rumen fill, body condition score, sex, type of cattle (dairy, beef), and distance travelled to the plant were not associated with the fecal prevalence of verotoxins or E. coli O157:H7. The prevalence of verotoxins in fecal samples of cull cows was associated with the source of the cattle. It was highest in cows from the auction market (52%) and farm/ranch (47%) and lowest in cows from the feedlot (31%). In rumen samples, the prevalence of verotoxins was 6.4% (95% CI, 4.2% to 9.4%), and it was 0.8% (95% CI, 0.2% to 2.3%) for E. coli O157:H7, and 0.3% (95% CI, 0.007% to 1.5%) for Salmonella.     

Valid methods: the quality assurance of test method development, validation, approval, and transfer for veterinary testing laboratories.

Third-party accreditation is a valuable tool to demonstrate a laboratory's competence to conduct testing. Accreditation, internationally and in the United States, has been discussed previously. However, accreditation is only I part of establishing data credibility. A validated test method is the first component of a valid measurement system. Validation is defined as confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. The international and national standard ISO/IEC 17025 recognizes the importance of validated methods and requires that laboratory-developed methods or methods adopted by the laboratory be appropriate for the intended use. Validated methods are therefore required and their use agreed to by the client (i.e., end users of the test results such as veterinarians, animal health programs, and owners). ISO/IEC 17025 also requires that the introduction of methods developed by the laboratory for its own use be a planned activity conducted by qualified personnel with adequate resources. This article discusses considerations and recommendations for the conduct of veterinary diagnostic test method development, validation, evaluation, approval, and transfer to the user laboratory in the ISO/IEC 17025 environment. These recommendations are based on those of nationally and internationally accepted standards and guidelines, as well as those of reputable and experienced technical bodies. They are also based on the author's experience in the evaluation of method development and transfer projects, validation data, and the implementation of quality management systems in the area of method development.