[The effect of granulosa cells on maturation, fertilization and cleavage in vitro]. / Der Einfluss von Granulosazellen auf die Reifung, Befruchtung und Furchung in vitro.

Addition of granulosa cells (GC) (1.10(6)/ml) affected maturation of non-ovulatory oocytes of cattle and caused delay in nuclear maturation (60.8 versus 35.3 or 37.1 percent). In vitro use of pre-incubated GC in the process of maturation led to higher rates of fertilisation (49.4 versus 25.2 or 21.0 percent) and of cleavage (29.6 versus 13.3 or 9.1 percent). No explanation has yet been found for different oocyte behaviours in response to use of fresh or pre-incubated GC. Transfer of seven embryos from maturation with GC as well as in vitro fertilisation and development to four recipients resulted in the birth of one female calf.

[Results of endocrinologic studies in culture media after coculture of bovine oocytes with granulosa cells]. / Ergebnisse endokrinologischer Untersuchungen in Kulturmedien nach der Kokultur von Rinderoozyten mit Granulosazellen.

The steroid hormones progesterone (P.), and testosterone (T.) were radio-immunologically determined in 108 medium samples, following co-culturing of bovine oocytes with granulosa cells. P. and T. values recorded from a control group were lower with significance than those recorded from co-culturing groups, that is 72 +/- 21 ng/ml and 264 +/- 84 pg/ml as compared to 208 +/- 138 ng/ml and 2,168 +/- 1,595 pg/ml in the oocyte plus fresh granulosa cell co-culturing group as well as 364 +/- 215 ng/ml and 825 +/- 233 pg/ml in the oocyte plus pre-incubated granulosa cell co-culturing group. These rises were accompanied by decline in maturation rate, increase in oocyte degeneration, and rises in the rates of fertilisation and segmentation.

[The inhibition of meiosis of bovine oocytes using biologic of synthetic inhibitors]. / Untersuchungen zur Hemmung der Meiose von Rinderoozyten mittels biologischer oder synthetischer Inhibitoren.

About 1,200 cumulus oocyte complexes were used in an attempt to investigate the influence in vitro of various biological or synthetic inhibitors on meiosis of bovine oocytes. The biological inhibitor used was a low-molecular protein fraction (OMI) of bovine follicle fluid. N6, O2-dibutyryl-cAMP (0.3-1 mol/l), hypoxanthine (4.0 mol/l), adenosine (0.75 mol/l), and nicotinamide (20.0 mol/l) were the synthetic inhibitors. The OMI-active lyophilisate caused inhibition by 34 percent after 24 hours of culturing. Meiosis was inhibited by 15-28 percent by the synthetic inhibitors. Eight transferable embryos have so far resulted from in vitro fertilisation of oocytes of extracorporal non-ovulatory maturation. Pregnancy was achieved in heifer recipients by tubal transfer.

Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes.

The influence of hardening of the zona pellucida of in vivo matured bovine oocytes on fertilizability was investigated. For the study, 163 preovulatory and 73 postovulatory oocytes recovered from superovulated heifers were used. The preovulatory oocytes, before they were used for in vitro fertilization, consisted of: 1) those cultured in vitro for 4 to 6 h to permit final maturation and 2) those incubated in the rabbit oviduct for 4 to 5 h to permit final maturation and induce hardening of the zona pellucida. A few oocytes served as a control of nuclear maturity and the zona pellucida solubility. Preovulatory and postovulatory oocytes were both inseminated in vitro using frozen-thawed, heparin treated and swim-up separated spermatozoa. Significant differences (P<0.01) were established between fertilization rates of cultured preovulatory oocytes (68.8%) and those incubated in the rabbit oviducts (42.9%), or those recovered from bovine oviducts (40.7%). It can be concluded that hardening of the zona pellucida distinctly influences the fertilizability of oocytes. This factor should be taken into account when considering the source of oocytes or the kind of treatment to be used for in vitro fertilization.

Serological detection of early pregnancy in cattle and partial characterization of a serum glycoprotein associated with early pregnancy.

Sera from heifers prior to artificial insemination (AI), 1-8 days after AI, and 7 days after embryo removal were investigated by crossed immunoelectrophoresis (CIE) by use of rabbit antiserum produced against bovine early pregnancy serum and intensively absorbed with non-pregnancy serum. One precipitation peak appeared in the alpha-globulin region when sera of non-pregnant heifers were under study. An additional peak could be demonstrated in the same region when sera of early pregnant heifers were investigated. By this method 91.5% of 71 sera samples were classified correctly to be pregnant or non-pregnant. The glycoprotein character of the above two serum components could be shown by binding to concanavalin A (Con A) in lectin affinity CIE. Relative molecular weights were estimated to be about 70,000 and 80,000 for the peptides of these two proteins applying sodium dodecyl sulfate polyacrylamide gel electrophoresis of precipitates cut out from CIE-plates. As shown previously close relation of this early pregnancy associated protein (EPAP) to the early pregnancy factor is supposed because of its characteristics and its ability to affect cellular immunity.

[The release of immobilized substances from Symplex capsules]. / Untersuchungen zur Freigabe immobilisierter Substanzen aus Symplexkapseln.

A new procedure of microencapsulation was studied with regard to substance release and quantification of diffusion processes on the capsule membrane. The permeability behaviour on the capsule membrane was especially studied in metabolites, which are essential for immobilized biological objects (i.g. preimplantative mammal embryos). Peptide and proteohormones, cyanmethemoglobin and proteins were enclosed in simple and multiple Symplex Capsules. All substances examined are able to pass the Symplex membrane. The speed of release is influenced by the size of the capsule, the ion force, temperature, concentration of immobilized substances as well as their linear and globular structur. Compared with simple capsules the release of substances from multiple capsules was delayed. Corresponding to the results found under the experimental design described the Symplex membrane can be considered as coating for the compartmentation of cells, that allows the passage of essential substances for the immobilized objects. The method of microencapsulation used and described has various ways of application.

[The effect of nutritional factors on the ruminal mucosa. 3. Condition of the mucosa after infusion of propionic acid, acetic acid and butyric acid]. / Untersuchungen über den Einfluss von Ernährungsfaktoren auf die Pansenschleimhaut

Three non-lactating cows (Deutsches Schwarzbuntes Rind) with large ruminal fistulas were fed coarsely structured food. Within a trial period of 21 weeks infusion periods lasting 3 weeks alternated with equally long control periods (K). During the 3 infusion periods, 8.4 mMol of propionic acid (P), 14.8 mMol of acetic acid (E) and 4,5 mMol of butyric acid (B) per kg liveweight per day were administered through the fistula, the total quantity being 19 litres of solution. In the periods K1...4 the ruminal fluid contained an average of 68 Mol% E, 19 Mol% P, 13 Mol% B (maximum of 10.25 mMol free fatty acids (FFS) per 100 ml, minimum pH 6.4). In the course of the 10 hrs of infusion the Mol percentages of the particular acids infused increased to 27% P (maximum of 11.14 mMol FFS per 100 ml, minimum pH 6.4) or 79% E (maximum of 12,99 mMol FFS per 100 ml, minimum pH 6.0 (5.5)) or 25% B (maximum of 10.34 mMol FFS per 100 ml, minimum pH 6.0 (5.5)). Infusions of E and B had the most pronounced effect on the ruminal mucosa compared with the K periods. All fatty acids increased the process of keratinization and decreased the size of cell nuclei in the stratum basale. As specific effect, P infusions produced a thickening of the lamina propria; B infusions caused a thickening of the stratum germinativum (proliferative effect) while e infusions led to a drastically reduced thickness of villi (antiproliferative effect) due to reductions in the stratum germinativum and the lamina propria. According to the morphological situation high specific mucosal function is suggested during the B-period. The mucosa appeared quite normal during all periods investigated, with the exception of the E period, where hyperkeratosis, atrophy and necrosis were observed in 34% of the sample. Changes in the state of the mucosa appeared as early as 1 week after the beginning of the respective trial periods. Keratin consolidation was the primary cause for chemically induced keratosis. The development of hyperkeratosis seemed to be favoured if low pH values occurred in the rumen in combination with small amounts of metabolites inducing proliferation, both representing synergistic factors.

[Studies on the effect of nutritional factors on the rumen mucosa. 2. Report. Morphological conditions after feeding with fattening rations in various physical forms]. / Untersuchungen über den Einfluss von Ernährungsfaktoren auf die Pansenschleimhaut 2. Mitteilung Morphologische Zustände nach Fütterung mit Mastrationen in unterschiedlicher physikalischer Form

3 groups of fattening cattle (DSR breed) were fed rations of nearly identical composition for a period of 395-455 days. The rations consisted of either crushed (group I), finely ground (group II) or finely ground and pelleted material (group III). Samples of the ruminal mucosa from 26 animals were investigated macroscopically, microscopically and histologically. All samples from animals of group I were found to be normal whereas clumping of the villi, hyperkeratosis, atrophy and loss of villi over 40%-50% of the surface of the ruminal mucosa were observed in the animals from group II, and, to a larger extent in animals of group III. The main reason for the mentioned changes of that kind are the very fine foodstuff particles, less than 0.05 to 0.5 mm in length, which form extraneous deposits in the mucosa or act as cement substances between neighbouring villi. This leads to hyperkeratosis because these particles prevent desquamation of the corneal cells and at the same time produce conditions of compression; these, in turn, result in a hardening of the newly formed stratum corneum. Hypertrophy of the undamaged villi was observed in animals of group III receiving the pelleted ration (thickness of villi group III: 243 mum as compared with group I:196 mum). The unfavourable morphological state of the ruminal mucosa in animals of group III was associated with a reduced rate of daily weight gains and increased food consumption.

[Studies on biotechnical ovulation synchronization in young cows. 1. Ovulation periods, ovarian findings and fertilization results after use of Metallibur, 750 IE PMS and 250 or 500 IE HCG]. / Versuche zur biotechnischen Ovulationssynchronisation bei Jungsauen 1. Mitteilung: Ovulationsperioden, Ovarbefunde und Befruchtungsergebnis nach Metallibur, 750 IE PMS und 250 oder 500 IE HCG

The following treatment was applied to 50 mature young sows in two experiments (22 and 28 animals) throughout 20 days: Turisynchron-Prämix (Turi.), 5 g/animal, and 750 IU PMS (Prolosan serum) 28 hours after Turi. In the first experiment. 250 IU HCG (Gonabion) were additionally injected to each of twelve animals 100 hours after Turi., while in the second experiment each of ten animals recieved 500 IU HCG 103 hours after Turi. The remaining animals of the two groups were used as controls. Inseminations took place 101 and 104 hours (fourth day) after Turi. in the first experiment and 125, 149, as well as 173 hours (fifth, sixth, and seventh days) after Turi. in the second. Onset of ovulation was brought forward to the sixth day after Turi. in response to 500 IU HCG by laparotomy performed in the mornings and evenings of the fourth through seventh days. Most of the controls and test animals with 250 IU HCG ovulated on the sixth or seventh day after Turi. Ovulation was stimulated by both HCG dosages, in comparison to the controls, which was established by slaughtering the animals between the seventh and twelfth days after Turi. The percentage of ovulations was higher among the test animals and that of ovarian cysts lower. Fertilisation of the second group was clearly better than that in the first where insemination had taken place two days prior to ovulation, that is too early. The latter results were secured by tubal douche and ovocyte tests.

[Studies on biotechnical ovulation synchronization in young sows. 2. Report: effect of laparotomy on ovarian findings after Zn-metallibur, 750 IU PMS, with and without 500 IU HCG]. / Versuche zur biotechnischen Ovulationssynchronisation bei Jungsauen. 2. Mitteilung: Einfluss von Laparotomien auf die Oxarbefunde nach Zn-Metallibur, 750 IE PMS, mit und ohne 500 IE HCG

A group of 72 gilts, aged between eight and nine months, were treated 20 days each by administration of 5 g Suisynchronprämix (Zinc Metallibur/Sui), followed by 24-hour treatment with 750 IU PMS (Prolosanserum). Fifty per cent of the group received 500 IU per animal of HCG (Gonabion) at 11 a.m., on the fourth day after Sui. All animals were artificially inseminated at 3.30 p.m. on the fifth day after Sui. and at 7.30 a.m. on the sixth day after Sui. Laparotomy was performed on 50 per cent of the HCG-treated and untreated animals in the afternoon of the sixth day after Sui. Animals with no recordable ovulation had to undergo another laparotomy in the morning of the seventh day. The above approach resulted in regrouping by four therapeutic categories: 1. HCG with laparotomy, 2. No HCG, 3. HCG with no laparotomy, 4. No HCG and no laparotomy. In the afternoon of the sixth day after Sui (51-56 hours after HCG) ovulation had begun in all 17 measurable animals of the first group, but only in one of 18 animals of the second. The animals were slaughtered between the seventh and twelfth days after Sui, and the following ovulation percentages were established: 100 per cent in the first group, 83.3 per cent in the second, 55.6 per cent in the third, and 72.2 per cent in the fourth. The animals that had been given HCG treatment (Groups 1 and 3) were found to be superior in terms of percentual ovulation to the untreated animals (Groups 2 and 4). However, Group 2 was the only group that had been exposed to the extraordinary stress of two laparotomies, and this should be borne in mind for evaluation. Ovarian cysts (more than 10 mm) began to develop on the eighth day on the laparotomised groups (1 and 2) and on the tenth day on the non-laparotomized groups (3 and 4). Cysts developed in 41.1 per cent of all animals in Group 1, 38.9 per cent in Group 2, 27.8 per cent in Group 3, and 22.2 per cent in Group 4. Therefore, cyst formation is thought to have been stimulated by laparotomy. Ovocyte tests suggested fertilisation of all animals in the first group. The embryonation rates of the second, third, and fourth groups are discussed with reference to the dates of insemination.

[Studies on biotechnical ovulation synchronization in young sows. 3. Report: ovulation onset, ovarian findings and results of fertilization after metallibur, 750 IU PMS, with and without 500 IU HCG with varied treatment time]. / Versuche zur biotechnischen Ovulationssynchronisation bei Jungsauen. 3. Mitteilung: Ovulationseintritt, Ovarbefunde und Befruchtungsergebnisse nach Metallibur, 750 IE HCG bei unterschiedlichen Behandlungsterminen

Fourty-eight gilts were treated with Turisynchron-Prämix (Turi.) and PMS (750 IU; 24-hours a. Turi.). One-half of the animals receaved additionally 500 IU HCG (fourth day a. Turi.). Performing treatments (Turi., PMS, HCG) either between 8 and 9 a.m. or 3 and 4 p.m. resulted in 2 experimental (HCG) and 2 control (without HCG) groups, each consisting of 12 animals. Double insemination took place according to treatment times at the fifth or the fifth and sixth day a. Turi. The experimental animals underwent laparotomy at the sixth day between 9 and 12 a.m., the conerols between 1 and 4 p.m. at the sixth or 9 and 12 a.m., at the seventh day a. Turi. Oviducts were flushed either at laparatomy or on slaughter to establish fertilization. From 24 experimental animals 20 ones had ovulated between 42-53 h p. HCG, and at slaughter 22 did so. The period of ovulation is mainly assumed near and immediately after 42 h p. HCG. In controls ovulation could be established in 3 of 15 animals laparotomized up to 152 h a. Turi. and in 8 of 9 animals laparotomized up to 168 h a. Turi. At slaughter there were in all 22 animals of the 2 control groups which had ovulated. In the rate of ovarian cysts (25-33%) and fertilized ova no remarkable differences were found between the groups.

[Studies of the influence of nutritional factors on the ruminal mucosa. 1. Structure and functional state of the ruminal mucosa after feeding of extreme rations and abrupt change in nutrition]. / Untersuchungen über den Einfluss von Ernährungsfaktoren auf die Pansenschleimhaut. 1. Mitteilung Strukturen und Funktionen nach Fütterung extremer Rationen und plötzlichen Futterwechsel

Different rations were used in successive experimental periods (Dried green feeds (I), fresh green feed from sugar beet tops (II), concentrates (III, IV), and maize silage (V), to test the effect they have on the structure and oxidative functions of the ruminal mucosa in cattle. Rations I, II, IV, and V were both energy and protein equivalent. Biopsy specimens from ruminal papillae were taken on the day when rations were suddenly changed and on the 21st and 22nd day of the feeding period; they were then investigated histologically and manometrically. It was found that some characteristics, (viz. the type and thickness of the stratum corneum, the thickness of epithelia, the size of cell nuclei in the stratum basale of the epitheliumas well as the state of the lamina propria and the oxygen uptake were subject to alterations depending on nutrition. Nutrition with energy-equivalent, but otherwise extremely different diets representing particular types of rations led to the development of different and quite specific functional states of the ruminal mucosa. All these functional states of the mucosa were found to be within the limits of normality but seemed to have a definitely more favourable functional effect in the case of rations I and IV than in the case of rations II and V. The feeding of concentrates (III, V) increased the energy intake to an amount of 6.6 kEFr, i.e. double that of the other rations, and brought about changes in quantitative parameters. These, in turn, indicated that proliferative and oxidative processes had been stimulated. Changes of this kind were accompanied by increases in the concentration of volatile fatty acids in the ruminal fluid which rose from a maximum of 9 mMol per 100 ml (ration IV) to 12.5 mMol per 100 ml (ration III). Immediately after any change in nutrition brought about by a change of rations, processes of adaptation occurred in the ruminal mucosa. A balanced state of the mucosa was again achieved after a period of not more than 3 weeks.