DOCTORAL (PhD) DISSERTATION THESES ANITA CSIBA MOSONMAGYARÓVÁR 2015 DOCTORAL (PhD) DISSERTATION THESES UNIVERSITY OF WEST HUNGARY FACULTY OF AGRICULTURAL AND FOOD SCIENCES MOSONMAGYARÓVÁR INSTITUTE OF ANIMAL SCIENCES AND BIOTECHNOLOGY Antal Wittmann Plant –, Animal-, and Food Sciences Multidisciplinal PhD School Head of Doctoral School Prof. Dr. Miklós Neményi University Professor, Corresponding Member of the Hungarian Academy of Sciences Imre Ujhelyi Animal Sciences Doctoral Programme Programme leader Prof. Dr. Ferenc Szabó DSc Consultant Dr. Elemér Gergátz CSc Senior Associate Professor THE DEVELOPMENT OF RAM SEMEN DEEP-FREEZING METHODS BASED ON THE RESULTS OF PHASE EXPERIMENTS Written by Anita Csiba MOSONMAGYARÓVÁR 2015 Table of Contents 1. INTRODUCTION ……………………………………………….4 2. MATERIAL AND METHOD …………………………………..6 2.1. Animals involved in the inspection…………………………….6 2.1.1. Rams…………………………………………………………...6 2.1.2. Buck……………………………………………………………7 2.1.3. Tests and experiments………………………………………...7 3. RESULTS AND DISCUSSION…………………………………..8 3.1. Examination the percentage change in motility%, the membrane and acrosome deformations, and the reproducing capacity during the cooling of ram semen cooled at 2-4 °C, based on data from autumn 2007……………………………………….....8 3.1.1. Motility% results of diluted seminal cooled at 2-4°C, year 2007…………………………………………………………………...9 3.1.2. Examination results of cell membrane and acrosome states in diluted and cooled ( 2-4°C) ram semen, year 2007 examination results………………………………………………………………..10 3.1.3. Successful insemination percentage using diluted semen cooled at 2-4°C, year 2007 results…………………………………11 3.2. Ram semen phase inspection of membrane and acrosome deformation examination during preparation, deep-freezing and thawing, based on spring 2008 data ………………………………11 3.2.1. CTC fluorescent staining procedure……………………….12 3.2.2. Light microscope inspections……………………………….14 3.3. Inspecting the effects of season change with regard to the suitability for deep-freezing, based on data from spring and autumn 2008………………………………………………………...17 3.3.1. Results of inspecting the effects of season change ……………………………………………………………………….18 2 3.3.2. Recommendations regarding the inspection of the effects of season change ………………………………………………………18 3.4. Year 2009 results ……………………………………………...19 3.4.1. Ram semen amount, year 2009 results……………………..19 3.4.2. The effect of thawing solutions containing decapacitating factors on the percentage of living cells and on decapacitation, as well as the application of incubation tests (at 39°C, 2h), based on year 2009 data………………………………………………………21 3.4.3. The inspection of decapacitation after thawing in different decapacitating solutions …………………………………………...22 3.4.4. Incubation experiment results, year 2009………………….24 4. NEW SCIENTIFIC RESULTS…………………………………27 5. SCIENTIFICT PUBLICATIONS AND LECTURES DEALING WITH THE SUBJECT MATTER OF THE DISSERTATION…………………………………………………...30 3 1. INTRODUCTION Until today, the deep-freezing of ram semen has not been unequivocally accomplished. Insemination with the help of thawed seminal deep-frozen using different techniques in artificial straw and pellet is in many cases only possible through surgical or semi-surgical ways, using the laparoscope method to get the semen into the horns of the uterus. Insemination carried out in such a manner can yield results if the seminal has 35-40% of motility%. However, for a successful cervical-uterine insemination carried out using the insemination catheter, seminal having 60-65% of motility% is required. Producing this is a very complicated task. It is enough if only a minor error occurs during deep-freezing, and the entire day’s work is wasted. When producing deep-frozen semen, one must pay attention to the exact composition of thawing solution, its proper quantity, making sure that pre-cooling is carried out in several steps; moreover, one must also exercise caution when adding the thawing solution containing the cryoprotective material, as well as make sure to observe the equilibration time, the deep-freezing speed and the thawing of the material at appropriate temperatures and in solutions of appropriate composition. The reproductive capacity of the spermatozoa, however, does not depend on the number of motility% alone. In many cases, the living, moving cells have already undergone the so-called capacity-like deformations, or acrosome reaction, the premature occurrence of which render them unsuitable for 4 insemination. Let us not forget, however, that it is crucial to insemination that these activities take place. During our investigations, the author examined these deformations with the help of the CTC (chlorine-tetracycline-hydrochloride) fluorescent dying procedure. This made it possible in the case of the samples examined to clearly establish the ratio of cells with an intact membrane that have undergone capacity-like deformations and the acrosome reaction. This thereby makes it possible for us to pinpoint the deep-freezing phase in which the spermatozoa get damaged, and also which steps need to be changed. During thawing, it becomes evident which composition of thawing solution is most suitable for reversing – that is, decapacitating – the capacity-like deformation that occurred during deep-freezing. Apart from the inspections above, we also carried out incubation tests (at 39°C, 2 h) in order to examine the vitality of ram semen thawed after deep-freezing. Apart from examination the deep-freezing methods, it is also crucial to raise, keep, feed, and regularly train proper rams in a stock that are capable of producing semen suitable for deep-freezing; furthermore, the quality of the acquired seminal must be continuously monitored and the results recorded so that during the investigation it turns out which specimen’s seminal is suitable for deep-freezing. We must also pay attention to the time of year, for example the autumn high season or the spring expletive season. In author’s experience, the animals produce seminal most suitable for deep-freezing in late autumn. In 5 case of the thawing solutions with different compositions, we examined their decapacitating effect. 2. MATERIAL AND METHOD During the examinations carried out at Pharmagene-Farm Ltd. between 2007 and 2009, the author examined the quantity and quality of spring and autumn ram semen, as well as its suitability for cooling down to 2-4°C and for deep-freezing with the involving of seven lacaune rams and a single buck. The amount of fresh semen acquired from the rams, the number of living cells in it, the number of living cells in the deep-frozen and thawed semen, as well as the ratio of spermatozoa that underwent capacitation-like membrane modification and acrosome reaction were determined in certain phases of the deep- freezing procedure. During the investigations, the efficiency of different decapacitating solutions was examined by living cell percentage determination, by examining the change in the ratio of cells that underwent capacitation-like deformation and those with intact membranes, as well as by employing incubation experiments, all of which served to examine the vitality of spermatozoa after thawing. 2.1. Animals involved in the inspection 2.1.1. Rams  299 6  4012  4045  4056  4245  23144  23386 2.1.2. Buck  Jimmy 001 2.1.3. Tests and inspections 1. Inspecting the percentage change in living cells, the membrane and acrosome deformations, and the reproducing capacity during the cooling of ram semen cooled at 2-4 °C, based on data from autumn 2007. 2. Ram semen phase inspection, membrane and acrosome deformation inspection during preparation, deep-freezing and thawing, based on data from spring 2008. 3. Inspecting the effect of the change of seasons on the suitability for deep-freezing, based on data from spring 2008 and autumn 2008. 7 4. Inspecting the effect of thawing solutions containing decapacitation factors on the percentage of living cells and on decapacitation, as well as application during heat exhaustion tests, based on data from 2009. 3. RESULTS AND EVALUATION 3.1. Inspecting the percentage change in living cells, the membrane and acrosome deformations, and the reproducing capacity during the cooling of tup semen cooled at 2-4 °C, based on data from autumn 2007 In autumn 2007, we examined seminal that was freshly acquired, diluted, and stored at 2-4°C. The investigations included the determination of the number of living cells using a light microscope, the determination of the ratio of cells that underwent capacitation-like deformation or acrosome reaction with an intact membrane using the CTC fluorescent dying method, as well as testing the seminal of different tups while using them for insemination. During this latter examination, we separately evaluated the success of insemination using the seminal acquired, which surprisingly yielded better results than the seminal used during the everyday routine. Insemination was carried out using the cervical-uterine method and a Milovanov type insemination catheter. The seminal used was freshly taken, diluted tup semen, as well as tup semen cooled at 2-4°C. Insemination was carried out twice a day: in the mornings and in the afternoons. 8 3.1.1. Motility% results of diluted seminal cooled at 2-4°C, year 2007 During the investigation, the living cell percentage was first determined immediately after the semen was acquired, then after 1 and 2 days of storage at 2-4°C. The results are included in the table below. Table 1:: Changes of motility % in ram semen on 2 - 4 °C storage Changes of motility % in ram semen on 2 - 4 °C storage Standard Average Ram’s ear number 4012 4045 4056 4245 deviation Storage Motility % Fresh semen 76,15% 77,00% 76,15% 77,27% 76,64% 0,58% 1st day 70,00% 67,50% 61,67% 71,00% 67,54% 4,18% 2nd day 55,00% 60,00% 55,00% 60,00% 57,50% 2,89% As a result of the investigation, it can be ascertained that out of the four samples examined and following 2 days of storage at 2-4°C, the based on the mean average of the living cell percentage in the seminal of two rams, namely that of #4045 and #4245, a result of 60% was measured, which means that generally speaking, the seminal is suitable for artificial insemination after two days of storage at 2-4°C. 9