The separation of bull spermatozoa was performed in a free-flow electrophoresis apparatus (Elpor Vap 11, Bender and Hobein). Fresh semen was washed twice and then the spermatozoa were suspended in the buffer medium for electrophoresis. The buffer medium was composed of 5 parts of triethanol-amine-potassium acetate buffer and 95 parts of 10% sucrose solution. The electrophoresis was carried out at 8-10°C and 50-60V/cm electric field.
Suspended bull spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber. The effluent from the bottom of the chamber was collected into 90 test tubes. The density of spermatozoa in each fraction was determined with the spectrophotometer.
Bull spermatozoa moved toward the anode and were separated into two main peaks. The motility of spermatozoa in some fractions before the first peak was very active, but in fractions after the first peak gradually decreased toward the second peak. The spermatozoa fractionated into 2-3 test tubes before the first peak were frozen in egg yolk extender and used for insemination. 87 cows of Japanese Black breed and 33 cows of Holstein breed were inseminated with the electrophoresed spermatozoa. A total of 79 calves was available to determine sex. 63.3% of the 79 calves were females. There was no evidence that the free-flow electrophoresis was useful for separation of X- and Y-spermatozoa of bull.

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Developmental potency of embryos produced by aggregating two to five 8- or 10-cell stage embryos collected from ddY strain mice was studied. Of 400 aggregated embryos produced, 393 formed morphologically normal single morulae or blastocysts in culture without degeneration of any blastomeres. Percentages of the aggregates which developed into normal morulae or blastocysts were not different among multi-embryo aggregates. However, developmental rate of the aggregates into the blastocysts tended to be slower as the number of embryos used for aggregation increased. At 12 hours after the start of culture, sizes of the embryos aggregated with 2-, 3-, 4- and 5-embryos were larger than a proportional size to the number of embryos aggregated. However, during incubation, sizes of the aggregates approached the size corresponding to the number of embryos aggregated. The morphologically normal morulae or blastocysts were transferred into the uteri of pseudopregnant females, and the females were autopsied on the 19th day of gestation. The percentage of implantation of five-embryo aggregates was significantly lower than that of two- to four-embryo aggregates (P<0.05). The percentage of the aggregates which successfully developed into live fetuses decreased as the number of embryos used for aggregation increased. Live fetuses derived from the aggregates were morphologically normal, though the body weight of the fetuses was lighter than that of fetuses obtained by natural mating. These results suggest that the developmental delay in culture or extremely increased size of aggregated embryo may affect the implantation and the embryo development during pregnancy.

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Voluntary dry matter intake (DMI) was measured for about 100 days after calving by using 21 lactating cows housed in the Hokuren Livestock Experimental and Training Farm. The cows were individually fed a total mixed ration consisting of TDN 73-74%, CP 15-17% and crude fiber 16% on a dry matter basis. Feed frequency was 3-7 times per day. Voluntary feed intake and milk yield were recorded daily, and milk fat and body weight weekly. The DMI averaged 23.8kg/day (maximun DMI 32.1kg/day) and 3.5% of weight (maximum 4.7%) in the average weight of 685kg and in the average 4% fat corrected milk (FCM) of 32.6kg. The DMI corresponded to 114% on average on a maximum DMI table in the 1978 ed. of NRC Standards. By using the data for 14 cows except for ones that suffered from heat stress, the significant regression equation for DMI (y) using two independent variables, body weight (x_w) and FCM (x_FCM), was as follows: y=13.1684+0.0068x_w+0.2213x_FCM. The equation gave a R² value of 0.59 and relative contributions were 21% for body weight and 79% for FCM. Moreover, by using the standard error of y, y and its 95% confidence limits were given between each body weight and FCM level. The maximum DMI values of the 1978 ed. of NRC Standard approximated 95% lower confidence limit values of y, and their % weight of maximum DMI was 0.2-0.8% units lower than those of y.

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The possibility of improving body fat percentage via living pig density was investigated. The selection response in backfat thickness of tested Landrace pigs (195 boars and 500 gilts) over four generations of selection, and the correlated response in carcass chemical fat percentage of 94 boars which were full siblings of tested pigs, were examined. The volume measurement apparatus for living pigs by air-displacement was made and the densities of 19 boars at about 90kg body weight were calculated by dividing the weight by the volume measured with this apparatus. The correlation between density and carcass fat percentage was estimated. The regression coefficients of backfat thickness on generations were -0.089cm for boars and -0.103cm for gilts. Carcass chemical fat percentage from the first to the fourth generation changed a little (23.7, 21.0, 22.9 and 22.8%), and the correlation between living density and backfat thickness decreased from 0.82 for the first generation to 0.55 for the fourth one. The coefficient of correlation between living density and carcass fat percentage was -0.740 (P<0.01), which was higher than that between living backfat thickness and carcass fat percentage (r=0.480, P<0.05). This suggested the effective improvement of fat percentage via living pig density.

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Fermented salami sausage (FS-1), mold-fermented salami sausage (FS-2) and FS-2 increased fat content (FS-3) or added with the effective component extracted from the mycelium of Penicillium miczynskii (FS-4) were prepared to study the changes in flavor and flavor related compounds during aging and drying process. The increase of free fatty acid and the production of cured meat flavors characteristic in mold-fermented salami sausage were noticeable on the surface of FS-2 and FS-3 and whole FS-4. It was suggested that this phenomenon of FS-4 was due to the action of the effective component of P. miczynskii. On the other hand, the increase of fat content in raw material showed no changes in the strength of cured meat flavor. All salami sausages had no significant difference in changes of ATP related compounds and organic acids. POV increased rapidly on the surface of FS-1.
To the contrary, it was restricted in all of FS-2, FS-3 and FS-4. From the results which were examined with lard emulsion, the restrictive action of fat oxidation was recognized in the culture solution, not in the effective component of P. miczynskii.

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In order to prepare BoLA typing reagents, screening and cluster analysis of alloantibodies against the BoLA were carried out using parous cattle sera and alloimmunized anisera for red cell typing. Sera from 571 parous cows (Holstein : 292, Japanese black: 133, Japanese brown: 72 and Japanese short-horn: 74) were tested by a two-stage microcytotoxicity dye exclusion assay against a panel of 100 lymphocytes (Holstein: 55 and Japanese black: 45). In total, the incidence of cytotoxic antibodies was 67%. The incidence of lymphocytotoxic sera increased gradually with parity (P<0.005). But there were no significant differences between 4 breeds (P>0.50). 153 alloimmunized antisera were tested against 40 lymphocytes. The cytotoxic antibodies were observed in 94% of these sera. Statistical analysis of the reaction patterns utilizing our own computer program revealed 8 clusters of sera which made it possible to detect 8 antigenic specificities. Each cluster consisted of sera from the following breeds; cluster 1 and 4: only Holstein, 7 and 8: mainly from Holstein, 3 and 5: mainly from Japanese black, 6: only Japanese brown, 2: all breeds. There were significant differences in the incidence of 6 antigenic specificities between Holstein and Japanese black. It was suggested that two of the 8 specificities, 7 and 8, showed a linear subgroup relationship. Using these 8 antigens, 55 Holstein were classified into 19 phenotypes, and 45 Japanese black were classified into 14 phenotypes.

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Proteins in tissue liquids can be analyzed by two-dimensional (2-D) electrophoresis and electroblotting techniques after running with 2-D electrophoresis. The developmental changes from colostrum to normal milk were studied by using 65 antisera of both human and horse origin, and then horse milk samples collected from mares within 1 hour to 2 weeks after parturition. A two-dimensional identification map of horse colostrum was first prepared and then the developmental changes in lactation period were observed by 2-D electrophoresis. The results were as follows: 1. Horse colostrum proteins were separated into 96 protein spots by 2-D electrophoresis. The colostrum proteins which could be identified by electroblotting-immunochemical staining techniques and by enzyme activity staining ones were comprised of the following 35 components; α-Lactalbumin (αLa), Prealbumin (PA), α_s1-Casein (α_s1CN), α₁-Antitrysin (α₁AT), α₁-Antichymotrypsin (α₁X), Albumin (Al), Gc-globulin (Gc), Prothrombin (FII), C-reactive-protein (CRP), Antithrombin III (ATIII), Aliesterase (Ali-Es), α₂-HS glycoprotein (α₂HS), Postalbumin (Xk), β-Casein (βCN), α₁-Acidglycoprotein (α₁AG), Transferrin (Tf), Lactoferrin (Lf), C9, Ceruloplasmin (Cp), Haptoglobin (Hp), IgG (T), α₁-Microglobulin (α₁Mi), C1q, Plasminogen (Pmg), C7, C4, C3c, IgG, IgA, Secretory IgA (SIgA), Fibrinogen (Fg), α₂-Macroglobulin (α₂M), Fibronectin (FN) and IgM. 2. As protein components stained with Coomassie Brilliant Blue R-250 (CBB), there were 72 protein spots in colostrum within 1 hour after parturition; after that, the number was decreased to 50, 31 and 20 spots in ones from about 9 to 14 hours, 24 to 72 hours, and 2 weeks, respectively. From these results and changes of immunoglobulin concentrations, milk within about 5 hours after parturition had characteristics of colostrum, after that, it changed a switch milk; ones passed 9 hours after parturition had already become a normal milk condition nutritionally. 3. Developmental changes from colostrum to normal milk could be observed by 2-D method. It means that 2-D electrophoresis can be applied for checking the quality level of colostrum and milks for colostrum bank. 4. Although protein components in colostrum were very similar to ones in plasma, five components of CN, La, Lf, SIgA and unknown milk components (M. C.) were observed as particular proteins in milk. Also, SIgA was detected in normal milk of 2 weeks after parturition, which showed a clearly different pattern as compared with IgG and IgM. 5. Polymorphisms of protease inhibitor (Pi-I), Es and Tf components could be detected in colstrum within about 5 hours after parturition, just as inserum.

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The influence of parameters on the age structures of a population (age class; 1-4 or 1-7 in nucleus males, 1-5 and 1-8 in nucleus females, 1-8 in base females), size of nucleus to base population (3, 6, 12%) and proportion of selected female via selection response in an open nucleus breeding system for beef cattle population were investigated, and selection responses in this system were compared with those in a closed system. Selection responses (response rates) per year were larger in smaller age classes and in lower proportions of nucleus males and females selected, but there was a negative relation between age class and proportion of selected females. The nucleus size of 12% was smaller in response rate than that of the other two. Response rates attained a steady state about 7 and 10 years after the beginning of the system, respectively, for systems of 4 and 7 age classes in nucleus males. Response rates in this system exceeded those in a closed system for any proportion of selected females, and these ratios increased, especially when the proportion of selected females rose above 80%. In accumulated selection response rates of nucleus population for 30 years after the beginning of the system, systems of the 4 age class in nucleus males exceeded those of the 7 age class by 33-36%, and systems of 60% in proportion of selected females exceeded those of 80% by 5-8%. Morever, ratios of nucleus to base population for accumulated responses were 1.11-1.13 and 1.21-1.25, respectively, for systems of 4 and 7 age classes in nucleus males. These results indicated that an open system is more efficient than a closed one for advancing the breeding of beef cattle population in Japan.

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The presence of gelatin in the mixed culture system of Clostridium 16-An and aerobes enhances the growth of 16-An and the production of collagenase. The molecular size of digested gelatin products in the mixed culture medium was investigated by gel chromatography. 1) Chromatography of the cell-free culture medium on an Asahipak 620 or Shim-pack 150 column clearly separated collagenase-digested products of gelatin (CD) from undigested gelatin, and enabled us to follow the gelatin-digestion process during cultivation. 2) Three peptides isolated from CD were estimated as having molecular weights of about 1, 000 (CD-1), 650 (CD-2) and 250 (CD-3), respectively. The CD-3 peak for the mixed culture medium decreased significantly during cultivation. 3) The amount of CD in culture, as estimated from the peak height on the chromatogram, rapidly reached a maximum level in 24hr of cultivation in accordance with the early stage of collagenase production. 4) The decreasing CD amount in cultivation stages longer than 24hr (of mixed culture) is attributable to the additional decomposition of amino acid residues such as Ala, caused by the growth of aerobes. This explains the high and long-life activity of collagenase production in the mixed culture system.

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1. Two in vitro studies were conducted to investigate the effects of various amount of nitrate in forages and of added nitrate on volatile fatty acids production with coarsely strained rumen fluid from sheep. 2. High-nitrate (0.56% nitrate nitrogen in dry matter), moderate-nitrate (0.22%) and low-nitrate (0.04 %) orchardgrass forages were prepared with three grades of nitrogen application. In experiment 1, each 20g (dry matter basis) of the forages packed into nylon bag was used as substrates for incubation trial. In experiment 2, 300, 100 or 0μg/ml of nitrate nitrogen as sodium nitrate at a final concentration was added to the buffer mixture along with low-nitrate (0.04%) substrate packed into a nylon bag. 3. The maximum accumulations of nitrite depending upon the amount of forage nitrate (experiment 1) and nitrate added (experiment 2) were observed within 12 hour after commencement of incubation. 4. Excess nitrate more than 0.22% in dry matter or 100μg/ml as nitrate nitrogen significantly (P<0.01) inhibited total volatile fatty acids production and increased the molar percentage of acetate, the oxidation product, with a decrease in molar percentages of the reduction products such as propionate and butyrate.

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This study was conducted in order to estimate genetic parameters on the muscle protein turnover rate in the Japanese quail, Coturnix coturnix japonica. 141 male progenies from the crosses of 19 males and 57 females obtained from a random-bred population were examined. Complete 24hr excreta collections were individually obtained for 3 days at 2 and 4 weeks of age, and the daily amount of excretion of Nτ-methylhistidine (Nτ-MH) was measured by High Performance Liquid Chromatography. Fractional rates (%/day) of synthesis and degradation were calculated by measuring the output of NT-MH in the excreta. Genetic parameters of the amount of Nτ-MH excretion and fractional rates of degradation (Kd) and synthesis (Ks) were estimated from sib analysis. Variation among dams within sires and among sire families on the amount of Nτ-MH excretion, the Kd and Ks were statistically significant at 2 and 4 weeks of age, respectively. The heritabilities(h2S+D)of the amount of Nτ-MH excretion, Kd and Ks were estimated as .49±.36, .55±.25 and .60±.26 at 2 weeks of age, and .46±.25, .65±.16 and .25±.21 at 4 weeks of age, respectively. The genotypic correlations between Ks and daily gain, fractional growth rate, and feed efficiency were positively high at both ages, and were higher than those between Kd and those traits. These values suggest that Ks is more effective on growth and feed efficiency than Kd. From these results, it was recognized that muscle protein turnover rates are controlled by genetical factors and are associated with growth and feed utilization. Additionally, these values suggest that individual information on muscle protein turnover rate may be available as a selection characteristic for growth and feed utilization.

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