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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Ronaldas Bilskis
IMMUNOCASTRATION OF YOUNG
AND MATURE BOARS
WITH IMPROVAC
®Summary of Doctoral Dissertation Agricultural Sciences,
Veterinary (02A)
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The work was done at the Department of Non-infectious Diseases Animal Reproduction Laboratory, Department of Infectious Diseases Patology Center, Department of Animal Husbandry Laboratory of Meat Characteristic and Quality Assessment of Veterinary Academy of the Lithuanian University of Health Sciences in 2008–2013.
Research Supervisor
Prof. Habil. Dr. Henrikas Žilinskas (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Veterinary – 02A).
Veterinary Science Council: Chairman
Prof. Habil. Dr. Antanas Sederevičius (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Veterinary – 02A).
Members:
Prof. Dr. Bronius Bakutis (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Veterinary – 02A);
Prof. Habil Dr. Zenonas Dobkevičius (Lithuanian Research Centre for Agri-culture and Forestry, Agricultural Sciences, Agronomy – 01A);
Prof. Dr. Gražina Januškevičienė (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Veterinary – 02A);
Prof. Habil. Dr. Aniolas Sruoga (Vytautas Magnus University, Science of Biomedicine, Biology – 01B).
Opponents:
Prof. Dr. Vytuolis Žilaitis (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Veterinary – 02A);
Assoc. Prof. Dr. Antanas Šarkinas (Kaunas University of Technology, Food institute, Technological Sciences, Chemical Engineering – 05T).
Public defence of doctoral dissertation in Veterinary Science Council will take place at the Veterinary Academy of Lithuanian University of Health Sciences auditorium of Dr. S. Jankauskas 10 a.m. on 1th of July, 2014.
Address: Tilžės 18, LT-47181 Kaunas, Lithuania.
The summary of doctoral dissertation was sent on 29th of May, 2014, according
to the confirmed address list.
The doctoral dissertation is available at the library of Veterinary Academy of Lithuanian University of Health Sciences.
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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA
Ronaldas Bilskis
JAUNŲ IR SUAUGUSIŲ KUILIŲ
IMUNOKASTRACIJA
PREPARATU IMPROVAC
®Daktaro disertacijos santrauka Žemės ūkio mokslai,
veterinarija (02A)
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Darbas atliktas 2008–2013 metais Lietuvos sveikatos mokslų universiteto Vete-rinarijos akademijos Neužkrečiamųjų ligų katedros Gyvulių reprodukcijos labora-torijoje, Užkrečiamųjų ligų katedros Patologijos centre, Gyvulininkystės katedros Gyvulių mėsinių savybių ir mėsos kokybės vertinimo laboratorijoje.
Mokslinio darbo vadovas
prof. habil. dr. Henrikas Žilinskas (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija – 02A).
Veterinarijos mokslo krypties taryba: Pirmininkas
prof. dr. Antanas Sederevičius (Lietuvos sveikatos mokslų universitetas, Vete-rinarijos akademija, žemės ūkio mokslai, veterinarija – 02A).
Nariai:
prof. dr. Bronius Bakutis (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija – 02A);
prof. habil. dr. Zenonas Dabkevičius (Lietuvos agrarinių ir miškų mokslo cent-ras, žemės ūkio mokslai, agronomija – 01A);
prof. dr. Gražina Januškevičienė (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija – 02A);
prof. habil. dr. Aniolas Sruoga (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01B).
Oponentai:
prof. dr. Vytuolis Žilaitis (Lietuvos sveikatos mokslų universitetas, Veterinarijos akademija, žemės ūkio mokslai, veterinarija – 02A);
doc. dr. Antanas šarkinas (Kauno technologijos universitetas, Maisto institutas, technologijos mokslai, chemijos inžinerija – 05T).
Disertacija ginama viešame Veterinarinės medicinos mokslo krypties tarybos posėdyje 2014 m. liepos 1 d. 10 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos dr. S Jankausko auditorijoje.
Adresas: Tilžės g. 18, LT-47181 Kaunas, Lietuva.
Disertacijos santrauka išsiuntinėta 2014 m. gegužės 29 d. pagal patvirtintą adresų sąrašą.
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INTRODUCTION
Castration (testicle removal) of males is performed for the reasons to avoid specific boar taint in meat and undesirable sexual or aggressive behaviour of animals. A boar taint is an offensive odor and taste of pork meat and its products that is made from sexually matured entire boars. Boar taint is caused by such compounds as androstenone produced in boars testicles and it‘s fragmentation products: skatole – a substance, that is produced in all pigs intestine, but has a tendency to be accumulated in entire boars fat (Zamaratskaia, 2004), indole (Rius and Garcia-Regueiro, 2001). Surgical boar’s castration is a matter of animal welfare. Scientific researches using physiological and ethological parameters have proved that surgical castration is a painful intervention, even when it is performed on very young animals. According to Statistics Department data in Lithuania in the year 2006 from 1.3 million pigs that are kept, 90.8 per cent boars were castrated. From 0.59 million castrated boars even 71.2 per cent were castrated without local aneasthesia. When more and more attention is given to the animal welfare, lately there are more discussions that surgical castration without the anaesthesia should be stopped (Stevenson, 2000). And finally the European declaration was accepted stating that from January 1st 2012 surgical boars castration should be performed only using a long term analgesia and /or anaesthesia methods and from January 1st 2018 surgical castration should be totally eliminated (European Declaration 2010). For these reasons an urgent search arises for surgical boars’ castration alternatives that would improve animal welfare without high expenses for farms or farmers. A number of surgical castration alternatives are known, but it all have advantages and disad-vantages. One of alternatives to lower stress and discomfort of animals is a surgical castration with local anaesthesia, but it needs labour costs and the use of pharmaceutics that makes the cost of procedure higher. Another alternative for surgical castration could be animal selection – when boars with lower androstenone amount in organism are chosen for the herd. But after lowering the amount of androstenone, the amount of anabolic hormones lowers too and it lowers entire boars and gilts grow effectively and makes their sexual maturation slower as well (Willeke and Pichner, 1989). Recently in stockbreeding a lot of attention is given to the use of sexated semen i.e. semen chosen by the sex. But for this kind of semen differentiation a big amount of semen is needed as during the
differ-6
rentiation a lot of cells die. It is said that just after significantly lowering the costs of obtaining sexated semen and after significantly improving the effectiveness of pigs’ self fertilisation by sexated semen it would be economically rewarding for swine breeding farms to use sexated semen (Zamaratskaia, 2004). A perfect alternative in our days for surgical castration is boars’ immunization against gonadotropin releasing hormone (GnRH), also called chemical or immunocastration (Bonneau et al., 1994; Dunshea et al., 2001; Turkstra et al., 2004). There are a lot of vaccines for immunocastration registered in the world: Equity® (Pfiser, South Africa) for fillies, Gona-Con® for deers, Repro-BLOC® (Pullman, USA) for cattles, Innosure® (Pfiser Inc.) and Improvac® (CSL, Australia) for boars and others (Kirkpatrick et al., 2011). Committee for Medicinal Products for Veterinary Use in Lithuania (CVMP) has done a research and came to conclusion that the benefit of medicinal product Improvac® is bigger than the risk it provokes when it is prescribed for all sexually matured boars to reduce boar taint and they recommended to give a marketing authorisation for it (EMEA). Improvac is an immunological product that has a mechanism of action similar to one of vaccine (it stimulates immune system to produce antibodies). The active substance of the product is an analogue of gonadotropin releasing hormone (GnRH) connected with protein carrier, produced from bacterium Corynebacterium diphtheriae. GnRH excluded from hypothalamus is the main hormone that sustains boar’s testicular functions, controls reproduction functions and repro-duction system progress. When vaccine is injected to boar, its immune system recognises synthetic GnRH as “foreign body“ and starts to produce antibodies that act against it. These antibodies connect to natural GnRH and suppress its impact. This vaccine reduces productivity of sexual hor-mones, including androstenone produced in testicles that is one of the main substances inducing boar taint. Besides, when the amount of sexual hormones is lower, liver can more efficiently clean the organism of boar from skatole and indole, reducing the amount of these substances in the body. The immuno-neutralisation of this hypophysis and gonads axis evokes temporal sterilisation for all mammals: it controls the symptoms of female rutting, depresses sexual and aggressive behaviour by males. Adjuvant (dextran produced from sugar) is also present in the composition of vaccine. Protein carrier and adjuvant provokes better response (EMEA). There are a lot of researches done in the world on performing immu-nocastration of young animals, but not so many data could be found in literature about the immunocastration influence on mature boars i.e.
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sexually mature and intensively used in reproduction or semen production. Meat of mature boars after they are used in breeding farms, pigs mating or insemination is also used for meat production – that is why before the slaughtering boars are castrated surgically or immunized against GnRH.
The aim of the study:
To evaluate the impact of immunocastration by commercial product Improvac® on young and adults, sexually matured boars.
The purpose of the study:
1. To evaluate the impact of immunocastration on intensity of grow, overweight and meat quality of young boars.
2. To evaluate the impact of immunocastration on testosterone concen-tration in blood, on libido, on semen’s quality and on biochemical indicators of adults/sexually matured boars.
3. To evaluate the impact of immunocastration on adults/sexually ma-tured boar’s testicles and accessory sex glands.
4. To evaluate the impact of immunocastration on meat quality of sexu-ally matured animals as well as on amount of indole and skatole in carcass.
1. MATERIALS AND METHODS
The work was done in two parts: “Immunocastration of young boars using Improvac® product“ and “Immunocastration of mature boars using Improvac® product“. First part of study was done in a breeding farm. The farm testing was done in natural conditions seeking to estimate the efectivity of immunocastration of young boars using commercial product Improvac®(Pfizer Animal Heath S.A., Belgium). Resarch was performed with 402 male piglets. The resarch scheme is shown in Fig. 1.1.
On the third day after the birth 204 piglets were castrated using surgical method with local aneasthesia and this group of animals was called Castrates. 198 left piglets (the “Improvac group”) were vaccinated with commercial Improvac® product two times: on 95th and on 138th day of living. Vaccination with Improvac® product was done following the instructions of the manufacturer: using a special vaccinator (person performing vaccination is protected from self-injection of drugs), prickling perpendicularly to the skin surface in the neck area just after the ear and
8 Castrates (n=204)
Surgical castration on 3rd day
1 VACCINATION 24th (weaning) day – weighting
60th day – weighting 95th day – weighting 138th day – weighting 181st day – weighting/slaughtering 2 VACCINATION II Improvac (n=198)
injecting 2 ml of product. The housing and feeding conditions for all piglets were identical. All animals were weighted in small groups on 24th (weaning) day, 60th, 95th, 138th day and on the day of slaughtering (181st) in the slaughterhouse.
Fig. 1.1. Scheme for the first part of the research/vaccination
Meat test. After slaughtering firstly the testicles of Improvac group
boars were separated and weighted. The boar taint in carcass was evaluated performing Express boiling test. When weighting carcass it‘s weight was set in kilograms. With lean meat meter (FOM – Fat-o-Meat’er) the muscularity of carcass was tested measuring fat and muscle thickness on the left side of pork carcass in these parts: in between 3th-4th lumbar vertebrae (fixating: Fat_1 thickness in mm), in between 3th-4th last ribs (fixating: Fat_2 thickness in mm). Also the thickness (mm) of carcass muscle was fixated i.e. carcass fat thickness was measured together with Fat_2 thickness in mm. Stated carcass quality i.e. carcass muscularity was evaluated in per cent. According to the muscularity carcases were divided into 6 classes (S, E, U, R, O, P). For chemical composition of meat and for physical and technological characteristics research 500 g meat samples were taken from the loin muscle by the last ribs. For the evaluation of indole and skatole concentrations fat samples (approximately 20 g) were
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taken. Samples in 0.5–1 h were delivered for research to LUHS VA Laboratory of Meat Characteristics and Quality Assessment. Samples were frozen to -80oC before the research. Such meat quality parameters were evaluated: pH, dry matter, colour (L*, a*, b*), water coherence, wate-riness, cooking loss, meat softness, back fats, proteins, ash content, oxyprolin, tryptophan, proportion of oxyprolin and tryptophan. Meat pH was set using pH-meter „INOLAB 3“ with contact electrode „Sensotix“; dry matter quantity – using automated weight for dry matter „SMO-01“ and drying samples in +105oC temperature; colour – measuring light of colour L*, rosiness a*, yellow colour b* with „Minolta Chromameter“; water coherence according to Grau & Hamm (1956); meat wateriness was evaluated by batch method keeping meat for 24 hours +4oC temperature in special bags (according to reduced sample weight in 24 h); cooking loss – by Shilling method, boiling vacuum packed meat in circulating water bath for 30 min in 75oC temperature; meat hardness – according to War-ner/Bratzler (1949); fat content – by Soxhlet method (Soxhlet, 1879); proteins – by Kjeldahl (Kjeldahl, 1883); ash content – combusting organic materials in 600–800oC temperature. Concentration of indole and skatole in fat samples were set by HPLC method following modificated Claus (1993) and Denhard (1993) methodology. Tryptophan content set by Spies & Chambers (Spies, 1967), oxyprolin − by Neuman & Logan methods (Neuman and Logan, 1950); index of meat proteins sufficiency – according to proportion of tryptophan and oxyprolin amino acids.
Research for the second part of current study “Immunocastration of mature boars using Improvac® product“ was done in commercial/pig breeding farm. Research was done on 9 investigative and 7 control mature boars from Danish Landrace breed. Taking into account the individual reaction of mature animal to a vaccine, during the research we were comparing all inspected boars‘ testosterone concentration in blood, ani-mals‘ libido, semen quality and parameters of biochemical blood charac-teristics in different research periods and variations of mentioned para-meters during the research time were analysed for each boar separately. At the end of the research when animals were slaughtered its testicles, appendage gonads and meat quality parameters were compared with mentioned parameters of slaughtered control i.e. nonvaccinated boars. Before the research all boars were used for reproduction i.e. collection of semen and sows insemination. Average age of the boars during the time of research was 28.33±9.71 months, weight – 234.63±69.39 kg.
10 Group A boars
(n=3)
Period 3 (4 weeks) 1 VACCINATION
Slaughtering of Group A boars
3 VACCINATION Slaughtering
of Group B
boars Slaughtering of Group C boars (n=3) Slaughtering of control group boars 2 VACCINATION Group C boars (n=3) Group B boars (n=3) Period 1 (4 weeks) Period 2 (4 weeks) Period 0 (3 weeks) The research lasted 15 weeks (Fig. 1.2). All tested animals were kept in individual pens, fed twice per day following the rations settled for breeding boars, that corresponds with standards applicable for quality compound feed in Republic of Lithuania, following the feeding norms, watered from automatic water troughs (Jeroch and others, 2004; Janciene, 2005). Animals were divided in four groups: CONTROL – control non-vaccinated boars; Group A – boars non-vaccinated two times (8 weeks and 4 weeks before slaughtering) and slaughtered; Group B – boars vaccinated two times (12 weeks and 8 weeks before slaughtering) and slaughtered in 8 weeks after the second vaccination; Group C – boars vaccinated three times (12 weeks, 8 weeks and 4 weeks before slaughtering) and slaugh-tered in 4 weeks after the third vaccination. Trial period was divided in parts: Period 0 – 3 weeks before the first boar‘s vaccination with Improvac product; Period 1 – four weeks in between the first and the second board‘s vaccination with Improvac product; Period 2 – four weeks in between the second and the third board‘s vaccination with Improvac product; Period 3 – four weeks after the third board‘s vaccination with Improvac
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product. After the research period all boars used in experiment were slaughtered, samples of testicle tissues were taken for histology test. During the trial period semen and blood of boars were taken once per week. Libido of boars that were newer used for natural pig mating was evaluated according to Ren (2009) in the 10 point system.
Tests of semen and spermatozoa
When ejaculation was taken first it‘s color and consistency were evaluated visually as well as it‘s volume in mililitres. 1 ml of undiluted ejaculation was poured into Eppendorf test tube to define the concentration of spermatozoa then the rest of ejaculation was diluted with BTS/An-drohep diluent and delivered to LUHS VA Laboratory of Animal Re-production for more detailed semen research. The sperm motility was evaluated by two methods: subjectively judging by the eyes and with the computer program for semen quality evaluation SCA® Sperm Class Analyzer (SCA® v.5.1-version, MICROPTIC S.L., Spain) fixating not less than 500 spermatozoa. Concentration of sperm was evaluated using phases contrast microscope (Nicon ECLIPSE 50i, Japan, 100×) with Goriajev camera (Pakenas, 1985).
Morphological evaluation of sperm heads was done colouring se-men with blue ink according to producer‘s instructions (Sperm Blue, MICROPTIC S.L., Spain). The following sperm head pathologies were registered: pier form, narrow basis, abnormal outline, undeveloped, ab-normal heads without tails, narrow, big, small ab-normal, short wide heads and spermatozoa with paracentrical middle part attachments. After evaluating the state of 500 spermatozoa a percentage of each pathology in a sample and a total percentage of sperm heads pathologies are counted. Hancock (wet test) method (Hancock, 1956) was used to evaluate sperm tails. 200 spermatozoa were evaluated. The following sperm tails infractions were registered: middle part alterations, tails twisted usually and under the head, double crooked tails and other pathologies: proximal and distal droplets, sperm head without tails, defects and changes of acrosomes, vacuoles. A percentage of each pathology in a sample and a total percentage of sperm tails pathologies as well as percentage of all others pathologies were counted. Test of sperm vitality is done colouring spermatozoon with eosin nigrosin and evaluating according to Dott methodology (1972).
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Blood test. During the research blood samples for biochemical and
testosterone tests were taken from ear vein to glass tubes without anticoagulant after each semen collection, at 9–10 a.m. Blood samples in 0.5–1 hour were delivered to laboratory. Separated blood serum was centrifugated for 5 min at 3000 rpm (rotation per minute) speed. 1 ml of blood serum from each sample was separated for tests and frozen till – 20oC temperature. Biochemical blood tests were performed with auto-mated computerized biochemistry analyzer SELECTRA Junior (Nether-land s, 2006). Reagents from Spinreact firm (Spain) were used for tests. Amounts of urea, glucose, creatine, calcium, magnesium, phosphorus triglyceride, general protein, alkaline phosphatase and HDL- cholesterol were identified in a blood serum. Concentration of testosterone in blood serum (ng/ml) was determined using computerized micro plate reader (Bio-Tek® Synergy HT, Bio-Tek® Instruments, Inc., USA, 2004) and TESTO-EASIA Kit (DIAsourse ImmunoAssays S.A., Belgium), following producer‘s instructions.
Histology tests of testicular tissue. Pathological-morphological tests on
testicles were done in Pathology Centre at of LUHS VA Department of Infectious Diseases. After boar‘s slaughtering its testicles and accessory sex glands in 0.5-1 hour were delivered to Pathology Centre. Test mate-rials were immediately evaluated macroscopically measuring size, weight, evaluating form, sectional view, colour and consistency. For tests 0.5 cm thick testicular tissue pieces were taken from front, middle and back side of a testicle. Collected pathology material for 24 hours was fixated in Bueno solution then was soaked for 3 days in 70° alcohol, changing alcohol each day to eliminate picric acid. Later dehydration was performed with alcohol in raising concentration: 70°, 80°, 96° and then pathology material was embedded in paraffin. Embedded pieces of tissues were cut with microtome in 4 µm thick cuts and glued on objective glass slides. Ready cuts were coloured with haematoxylin eosin. Coloured cuts were covered with covering glass slide and lued with Canada balsam then it was exposed on light microscopy (Lauruseviciene and Smaliukiene, 2008).
Statistical data analysis. Statistical data analysis was done with SPSS
statistic package version No 13 (SPSS for Windows 13.0, SPSS Inc., Chicago, IL, USA, 1989–1999). Collected data was processed with „Mic-rosoft Excel“ (Mic„Mic-rosoft Office Excel, 2003) program. When analysing data firstly its description was done using descriptive statistics (arithme-tic average, standard deviation, minimum and maximum values). Data was analysed using mono-factorial analysis method. The importance of
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elements was checked hypothesizing that these factors have no influence on chosen variable. Elements with lower meaning than settled limit (P=0.05) were treated as the ones having influence. Differences between groups were analysed using LSD multiple comparison method. Data was treated as statistically reliable when * P≤0.05; ** P≤0.01; *** P≤0.001.
2. RESULTS OF THE INVESTIGATION
2.1. Affectivity research on young boars immunocastration
with Improvac® product
During the research 58 piglets died for various reasons i.e. dead rate was 14.43%, but not higher than usual percentage (10-15%) of piglets dead rate on a farm. Current research results have shown that on a day of weaning boars from the surgically castrated group weighted 0.91 kg in average, that is more than boars from the Improvac group (P≤0.001) (Fig. 2.1.1.). In a 95th and 138th day of breeding boars from the surgically castrated group weighted 1.01±0.93 kg and 7.59±1.66 kg respectively and that was more than boars from the Improvac group (P≤0.001). But at the end of the current research, on a day of slaughtering, boars from the Improvac group weighted more (P≤0.01) in average 2.02±3.93 kg. Live and carcass weight of Improvac group boars was bigger 2.02±3.93 kg (P≤0.01) 0.93±0.42 kg (P≥0.05) respectively if compare with surgically castrated animals weight.
Fig. 2.1.1. The dynamics in research groups boars‘ weight changes during all the period of its‘ live on different weighting days
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Average weight gains in the period from 60th day before the first vaccination and from the first vaccination till the second vaccination were bigger in castrated animal group 0.08±0.04 kg and 0.15±0.03 kg (P≤0.001) respectively. But after the second vaccination till the day of slaughtering the average day weight gains in Improvac group of boars was 0.24±0.1 kg bigger if compare with surgically castrated animals (P≤0.001).
Carcass tests results have shown that vaccinated animals‘ meat was leaner, muscle thickness and percentage of a lean was bigger. Fat_1 thickness of castrated animals was 3.44±1.26 mm, fat_2 thickness was 2.02±0.91 mm i.e. bigger than vaccinated animals‘ carcasses (P≤0.001). Castrated animals‘ carcasses muscle thickness was slightly smaller (0.04±0.38 mm) if comparing with vaccinated animals (P≥0.05). Carcass percentage of lean was 1.93±0.71% bigger in vaccinated animals (P≤0.001). In Improvac boars group there were more carcasses that ful-filled higher quality standards for muscularity class SEUROP (Fig. 2.1.2.). 6.7% from all Improvac group boars‘ carcasses were accredited to muscularity class S when none of carcasses from castrated animals‘ group corresponded to class S. 72.97% of Improvac group carcasses and 58.39% of castrated boars group carcasses were accredited to class E. 34.9% and 20.27% of carcasses respectively were accredited to class U.
Fig. 2.1.2. Research boars groups carcasses classification according to muscularity classes SEUROP (n=297)
When smell tests i.e. boiling tests were done in a slaughterhouse there were no samples detected with boar taint. Laboratory tests on indole and skatole amounts in carcasses samples have shown that amounts of these substances were not higher allowed norms. But anyway in castrated
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animals carcasses indole as well as skatole amounts were lower 0.19±0.05 μg/g and 0.02±0.01 μg/g respectively if compare with vaccinated animals (P≥0.05).
2.2. Affectivity research on mature boars immunocastration
with Improvac® product
During the all research period animals taking part in the current research were clinically healthy and no health problems were noted during the mentioned period. Just for one boar a slight swelling was noticed at the injection site.
Tests on immunocastration effect on testosterone amount in blood of mature boars have shown that testosterone concentration in the Period 1 was 1.58±0.72 ng/ml higher than in the preparation period (P≤0.001) (Fig. 2.2.1.). In the Period 2 testosterone concentration was recorded as 2.12±0.6 ng/ml (P≤0.001) and in the Period 3 even 2.95±1.13 ng/ml (P≤0.001) lower than in the preparation period. In the preparation period animals‘ libido was optimal and evaluated to 8±0 points. After the first injection of Improvac product the activation of animals’ sexual attraction was noticed: bigger salivation, faster jump on jackstraw/mannequin, faster ejaculation start time and shorter ejaculation itself. Sexual behaviour of animals in this period was evaluated as hypersexual and 10 points were given (Fig. 5.). In Periods 2 and 3 animals‘ libido was 1.47 (P≤0.001) and 5.04 (P≤0.001) points less respectively if compare with preparation period.
Fig. 2.2.1. Testosterone concentration in boars’ blood serum and animals‘ libido during different research periods
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Sperm vitality during the research period has varied insignificantly. The biggest amount of live and motile spermatozoa was noticed during the preparation period. At the end of the current research i.e. in the Period 3 sperm vitality has lowered by 7.37 per cent (P≤0.001) if compare with the preparation period and subjectively set sperm motility has lowered by 14.19 per cent (P≤0.001). Subjectively and objectively noticed sperm motility was correlating positively in between (r=0.244; P≤0.01). The biggest average total volume of all groups boars ejaculate was set during the Period 1. At the end of the current research average volume of ejaculate was 96.44±21.62 ml lower (P≤0.001) if compare with the preparation period. The smallest average concentration of spermatozoa was noticed also during the Period 3 and it was 0.006±0.06 milliard/ml lower (P≤0.001) if compare with the preparation period.
Mature boars immunocastration using Improvac product had influence on a total growth of pathological spermatozoa in ejaculate (P=0.004) too. Average total per cent of healthy spermatozoa has lowered by 27.54 per cents during the research period. Total per cent of sperm heads‘ pathologies, pathological sperm tails and other pathologies of spermatozoa in ejaculate has risen in average by 1.78 per cent (P≤0.001), 5.65 per cent (P≥0.05) and 10.37 per cent (P≤0.001) (Fig. 2.2.1.) respectively during all current research.
Fig. 2.2.2. Variation of separate spermatozoa pathologies groups during current research periods.
Immunological castration using Improvac product had no significant influence (P≥0.05) on biochemical blood indexes by mature, sexually adult
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and already in use for reproduction boars. Average meanings of bioche-mical blood indexes in the preparation period as well as in the research period were in between the limits of the physical standard.
After immunocastration impact analysis for each boar’s group separa-tely, similar tendencies of testosterone concentrations, libido and semen quality variation during current research were defined independently from the vaccination scheme. In the preparation period in Group A boars‘ blood testosterone concentration was 4.14 ng/ml in average. The peak of testosterone concentration amount in research boars‘ blood was found after two-three weeks after the first injection of Improvac product i.e. in the 5th -6th week of the research period. The lowest testosterone concentration in blood was found in the 11th week of the current research, just before the slaughtering of animals and was in average 0.47 ng/ml (P≤0.001) lower if compare with the preparation period (Fig. 2/2/3).
Fig. 2.2.3. Group A boars‘ testosterone concentration variations in blood during the research period (n=3)
Boars’ vaccination with Improvac product had no statistically impor-tant influence on sperm motility (P≥0.05). It was in average 51.04% of healthy spermatozoa in ejaculates of Group A boars in the preparation period (1st-3rd week of the current research). At the end of the research i.e. in the 11th week of the current research, percentage of healthy spermatozoa was 3.7% lower in average (P≥0.05). The biggest part of other sperm pathologies was spermatozoa with proximal (40.92%) and distal (57.25%) cytoplasm droplets.
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Fig. 2.2.4. Variation of testosterone concentration in blood serum of Group B boars during the research period (n=3)
Testosterone amount of Group B boars during the preparation period was 3.03 ng/ml in average. On the 5th week of the current research the biggest average amount of testosterone in boars‘ blood was found; it was 54.8% bigger than the average testosterone amount during the preparation period (P≤0.05). Testosterone amount in blood of animals taking part in the research during the Period 3 (12th-15th week of the current research) was found even 83.2% lower if compare with testosterone amount in blood of these animals during the preparation period (P≤0.001) (Fig. 2.2.4). Direct connection between the testosterone concentration in blood serum and the sexual activity of animals taking part in the current research was noticed (r=0.565; P≤0.001) too.
Morphologically healthy, unaltered spermatozoa in ejaculates of Group B boars during the preparation period were 59.33±4.13% in average. But in the last week of the current research in mentioned group boars’ ejaculates it was by 17.5% less (P≥0.05) of healthy spermatozoa.
Testosterone amount of Group C boars during the preparation period was 3.25±2.25 ng/ml in average. Similar to already mentioned boars groups, the tendency of remarkable increase of testosterone amount in blood was noticed in the second week after the first Improvac product injection. During the 5th week of the current research the peak of testo-sterone amount in boars blood was noticed – 6.52±2.66 ng/ml. After the 5th week of the current research the consistent diminution of testosterone concentrations in animals‘ blood was observed till the minimum meanings (0.081 ng/ml or 0.036 ng/ml) in the last weeks of the current research (Fig. 2.2.5.).
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Fig. 2.2.5. Variation of testosterone concentration in blood serum of Group C boars during the research period.
Immunocastration using Improvac product for Group C boars had no big influence (P≥0.05) on sperm motility. But the average sperm vitality in Group C boars during the preparation period was 94.94%, when at the end of the current research, in the last week it was 14.77% lower if compare with the peparation period (P≤0.001). In the peparation period in ejaculates of Group C boars it was in average 77.83% healthy, i.e. morphologicaly unchanged spermatozoa. During the research the amount of spermatozoa with tail pathologies as well as other spermatozoon pathologies was increasing progressively in ejaculates. During the last week of the current research in ejaculates of Group C boars it was 47.32% more pathological spermatozoa if compare with preparation period (P≤0.001).
2.3. Research of immunocastration influence on testicles and accessory sex glands
Results of the current research of immunocastration influence on mature board‘s testicles and appendage gonads have shown that the average weight of control group boars‘ testicles was 700.14±82.69 g big-ger than of Group C boars (P≤0.01) and 529.14±60.33 g bigbig-ger than of Group B boars (P≤0.001). Just the average weight of Group A boars‘ testicles had a very small difference (P≥0.05) if compare with control group boars‘ testicles weight. Amount of testosterone in blood of research boars had a positive correlation with the total testicle weight (r=0.615; P≤0.05) and with the amount of skatole found in fat (r=0.641; P≤0.01) and in omentum fat (r=0.621; P≤0.01).
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In a histology test it was stated that in testicles of control group boars a usual spermatogenesis takes place with appropriate cells for the cycle (Fig. 2.3.1). In interstitial tissue one can see numerous collections of Leydig cells. In Group A boars‘ testicles histologically no alterations in spermatogenesis cycle were found.
Fig. 2.3.1. Histological image of a healthy and functioning testicle cross section
Normal reproductive cycle in an interstitial tissue of a Leydig cell that is responsible for testosterone production (H&E, 100×).
In testicles of Group B boars a spermatogenesis cycle is disturbed, there are no spermatids, just stem gametes are left, gigantic multinucleated cells and protein mass accumulations are observed in tubular centre. Sperm granuloma was found in one testicle. Less Leydig cells are in connective tissue, focal infiltrations of lymphocytic and granulocytic cells are found (Fig. 2.3.2).
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Fig. 2.3.2. No gametes are left, Sertoli cells are observed
There are Leydig cells, eosinophils, lymphocytes, and plasmocytes in interstitial tissue. Protein mass accumulations are observed in some tubulars (H&E, 200×).
In all testicles of Group C boars a chronic interstitial lymphocytic inflammation and distinct signs of cell degeneration were noticed (Fig. 2.3.3).
Fig. 2.3.3. Testicle atrophy with obvious calcinosis (H&E, 100×)
After analysing the impact of Improvac product for appendage gonads it was stated that vaccination of mature boars had no statisticaly reliable negative impact for appendage gonads (P≥0.05).
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2.4. Research of immunocastration influence on boar taint and carcass quality
Current research results have shown that vaccination of mature boars with Improvac product had no significant impact on indole concentration, especially in omentum fat (P≥0.05), but was important for another impor-tant boar taint component – skatole, i.e. its concentration in fat as well as in omentum fat. Skatole concentration in fat of Groups A, B and C boars was 0.36±0.31 μg/g (P≤0.01), 0.57±0.31 μg/g (P≤0.01) and 0.62±0.31 μg/g (P≤0.01) respectively less if compare with control group. Skatole concentration in omentum fat of Groups A, B and C boars was 0.03±0.43 μg/g (P≤0.05), 0.24±0.43 μg/g (P≤0.05) and 0.64±0.43 μg/g (P≤0.05) respectively less if compare with control group. Skatole concentration in fat and omentum fat was as well correlating positively in total testicles weight r=0.58 (P=0.019).
During the research it was stated that colour indicators in meat of Group C boars were the highest (P≥0.05) (Fig. 2.4.1). Meat colour L was correlating negatively with total testicle weight (r=–0.524; P=0.037) and skatole amount in omentum fat (r=–0.504; P=0.046), but it was correlating possitively with back fat (r=0.539; P=0.031). Meat colour b was corre-lating possitively with back fat (r=0.560; P=0.024).
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3. DISCUSSION
Surgical castration of boars is used in practice for reasons to avoid objectionable sexual or aggressive behaviour by males and appearance of boars taint. In our days inside and outside EU various alternatives for surgical castration are already used widely. One of alternatives is boars immunization against gonadotropin releasing hormone (GnRH), also called chemical or immunological, i.e. immunocastration (Bonneau et al., 1994; Dunshea et al., 2001; Turkstra et al., 2004). With current research we were seeking to evaluate impact of immunocastration by commercial Improvac® product for young as well as for mature, sexually adult boars, i.e. in use for reproduction.
In the first part of the research the investigation was done in natural farm conditions, where we were seeking to evaluate immunocastration impact on young boars’ breeding intensity, overweight and meat quality. During the research we have found that animals were tolerating well the injection of this product and we have proved the results by foreign scientists that animals are tolerating well this vaccine and that vaccine does not evoke any significant negative side effect (Dunshea et al., 2001; Zamaratskaia et al., 2008; Pauly et al., 2009; Gispert et al., 2010; EMEA).
Meat is the most important source for animal protein. Physical and chemical qualities as well as biological value of different types of animals‘ meat is not equal, but according to Statistics Department data, pork is still one of the most popular type of meat used by citizens of Lithuania. Pork is popular because pigs are prolific, quickly become mature, obtained carcass output is big and comparingly the price for pork is the smallest. Con-sumers evaluate the most a meat that is not fat, juicy and has high biolo-gical and culinary qualities (Jukna and others, 2007). It is said that because of an anabolic hormonal effect secreted by testicles, entire boars absorb feed more effectively and because of this reason the meat is more lean (Xue et al., 1996). But androstenone, skatole and indole that accumulate in entire boars’ fat adds to meat unpleasant boar taint and meat becomes unacceptable for use (Claus et al., 1994). When it is more and more spoken about animal welfare a motivation to resign from surgical boars‘ castration totally is increasing and that is why boars’ immunocastration by immu-nization against gonadotropin releasing hormone (GnRH) is more and more used in the world. Scientific research done in the world with young boars, when the first vaccine injection is done in its 8th-18th week and vaccination is repeated when 4–6 weeks are left till intended slaughtering,
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has proved that young boars‘ immunocastration has signifycantly lowered the number of aggressive and sexual behaviour cases i.e. poking, fighting among themselves and riding on each other in a herd (Brewster and Nevel, 2013). When aggressive and sexual activities of animals are lower, they eat more peacefully, they spend more time standing calmly or lying. In this way immunocastration of young boars, also influencing animal welfare as well, indirectly influences more effecttive consumption and uptake of boars feed, bigger overweight (Brewster and Nevel, 2013), as well as leaner the carcass itself (Pauly et al., 2009). It is said that energy value of pigs when standing or in resting state is very high (Noblet et al., 1993). The tests done during our research have proved the results achieved by foreign scientists: vaccinated boars‘ day overweighs were bigger, on a day of slaughtering vaccinated boars weighted more than castrated boars and meat of vaccinated boars was leaner (P≤0.05). The results of animal weighting on different life stages had shown that on a day of weaning, what was on average the 24th day of life, surgically castrated boars weigh-ted a bit more on average than Improvac group boars (P≤0.001). This can be explained by bigger activity of entire animals and bigger loss in weight because of this. Castrated animals jump less on each other and loss less weight than entire, sexually active boars (Albrecht et al., 2012). Animal weighting results on 95th and 138th day of boars’ growth have shown that boars from surgically castrated group weighted more than boars from Improvac group (P≤0.001). This difference in weight again can be ex-plained by testosterone amount in blood that increases with age in Improvac group boars and that provokes males’ aggressive and sexual be-haviour and bigger loss in weight. And just in the last period of our current research, i.e. in the period after the second vaccination till slaughtering, when GnRH is strongly blocked and secretion of gonadotropin hormone is getting lower, Improvac group boars have gained more weight and on a slaughtering day they weighted in average 2.02±3.93 kg more than boars castrated on their first days of life (P≤0.05). Similar research results were obtained by other researchers too (Millet et al., 2011). It was stated that concentration of testosterone is getting lower after the injection of the second vaccine dose (Claus et al., 2007; Albrecht et al., 2012). And that other appearances are connected with lower testosterone secretion too: decreased aggressive and sexual activity (Giersing et al., 2000).
Research results on carcass quality itself have shown that vaccinated animals meat was leaner, layer of muscle was thicker and muscularity percentage was higher (P≤0.001). Carcass quality results according to
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muscularity classification SEUROP have proved that there were more carcasses in Improvac boars group that fulfilled the requirements for higher quality according to muscularity classification SEUROP. Diffe-rence in percentage of carcass muscularity between boars from castrates and Improvac groups is because of higher percentage of Improvac group boars‘ carcass in ham and pork shoulder areas, but not because of per-centage from loin muscle. Muscles responsible for movement are deve-loping in the earlier stages of life. As the second vaccination dose reduces functioning of anabolic hormones in the period of late phase of develop-pment, it is believed that this makes bigger impact on development of loin muscle (longisimus dorsi) than of muscles from ham or shoulder areas (Pauly et al., 2009).
There is little data in literature that could prove immunocastration makes influence on separate meat quality parameters. More detailed meat quality tests, done in Meat Research Laboratory during our current re-search have also shown not so many differences on separate meat quality parameters between surgically castrated and vaccinated boars‘ meat. It was noted that percentage of meat wateriness, a-colour rosiness, b-colour yel-low tint and ashes was bigger in Improvac group boars‘ meat, but per-centage of back fat and amino acid oxyprolin mg/percent was found higher in surgically castrated animals’ meat (P≤0.05). With the results of our current research we have just proved statements by other authors, saying immunocastration has an influence on meat lightness, wateriness, amount of ashes (Silveira et al., 2008), amount of back fat (Gispert et al., 2010), but for carcass pH, boiling loss, boiling coherence and other parameters it has no significant impact (Pauly et al., 2009; Škrlep et al., 2012).
After performing boiling test in slaughterhouse there were no samples found with a board taint. Similar results were obtained by other researches too. Scientist Pauly with co-authors (Pauly et al., 2009) after investigating 52 vaccinated boards has stated, that testicles of one of it was even bigger and heavier than testicles of entire boars. This scientist thinks that size of testicles is also very dependent on the total weight of a board‘s carcass and he suggests to judge about the affectivity of vaccination not just from the size of testicles, but from the total carcass weight and boar taint substan-ces: androstenone, skatole and indole concentrations in fat as well. In our current research on surgically castrated animals we have found less indole as well as skatole: 0.19±0.05 μg/g and 0.02±0.11 μg/g respectively, if compare with vaccinated animals‘ carcass (P≥0.05), but the amounts of these substances were below the sensory threshold. We think that
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vaccination of young boars with Improvac product while reducing amount of hormones in testicles made the metabolic clearance of indoline substances faster , i.e. it lowered skatole and even indole concentrations in fat tissue (Dunshea et al., 2001; Zamaratskaia et al., 2008; Pauly et al., 2009; Gispert et al., 2010). The results of current research let us claim that immunocastration with Improvac product for young boars made better feed conversion and influenced bigger overweight, increased muscularity in carcass and reduced concentration of skatole and indole in carcass that make unpleasant meat smell to minimum values and this is the reason why this castration method could be effectively used in Lithuanian farms.
Veterinarian was examining research animals during all the current research time i.e. 15 weeks investigating mature boars and no significant health problems have been noted during the research time. This was proved by other scientist researches in the world too that young boars as well as mature ones tolerate Improvac product perfectly (Kauffold et al., 2010; Agudelo-Trujilo et al., 2011) and almost the only possible side effect is swelling at the vaccine injection site (EMEA).
As a normal reproduction activity of male we usually consider: his libido, production of spermatozoa and semen itself, ability to perform a sexual intercourse, to ejaculate and to fertilize the female. These sexual male functions are strictly controlled by his central nerve system, GnRH, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that stimulate and by feedback activity control all hormones involved in sexual system, especially androgen testosterone activity. Testosterone is neces-sary for normal testicle activity i.e. production of spermatozoa, it initiates the development of secondary traits, its activity influences sexual reflexes functions and male sexual activity libido depends very much on the amount of testosterone. In mature male organism testosterone affects muscles, blood plasma, and metabolism of plasma proteins (Holdstock et al., 1982; Settle et al., 2001; Risbridger et al., 2003; Hafez and Hafez, 2000). With immunocastration GnRH as well as the release of gonadotro-pin hormones are blocked and because of that normal activity of testicles and appendage gonads is disorganized and pathological processes evolve in testicles. In this way immunocastration indirectly influences also the quality of semen itself: its quantity as well as quality parameters.
In literature sources a variation of testosterone concentration and its peaks in boars‘ blood serum during a day are usually mentioned, but there is no unanimity on what time of a day the testosterone concentration in boars blood serum is the smallest and on what time it reaches the peak. In
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our current research for the reason to reduce as much as possible the influence of a day time factor to testosterone concentration, all boars‘ blood samples during all the current research time were taken at the same time: at 10 o‘clock in the morning, just after the semen collection. During the preparation for the research period, i.e. in nonvaccinated boars‘ blood serum the testosterone concentration found in individual boars‘ blood serum has varied from 0.91 ng/ml till 6.43 ng/ml. Boundaries of testoste-rone concentrations in separate boars‘ blood serum indicated in foreign literature are also extensive (1.11–8.08 ng/ml) (Albrecht et al., 2012). Tests on immunocastration impact on testosterone amount in mature boars blood have shown that testosterone concentration in all groups of the cur-rent research animals‘ blood serum after the first Improvac injection has increased but later it has been decreasing gradually (P≤0.001). A peak of testosterone concentration in boars‘ blood serum was stated after two-three weeks following the first Improvac injection, i.e. on 5th-6th week of current research period and after the consistent reduction of testosterone amount it was noticed to drop even till minimal meanings (0.081 ng/ml) or even below setted limits in the last weeks, i.e. on 14th-15th week of the current research and not depending on if boars were vaccinated two or three times. This can be explained that after the first injection of vaccine dose a gonadotropin releasing hormone (GnRH) was activated and it had influenced the activity of testosterone concentration production in Leydig cells, in testicles. After two-three weeks after the vaccination in animal’s organism the antibodies against GnRH start to be produced and its activity is gradually getting weaker. LH activity is weakening and that influences Leydig cells function slowdown, i.e. weaker production of testosterone and all sexual functions. It was stated that testosterone decrease is not con-nected with atrophy or degeneration of germinal cells. It is concon-nected with Leydig cells affection, diminution of its volume as well as with decreased production and a release of testosterone (Sernienė et al., 2005). After repeated injection of Improvac product the amount of antibodies in animal‘s organism increases even more, GnRH is blocked stronger and boars sexual functions weaken even more. In literature still not so many data could be found about the immunocastration impact for mature boars that are already used in reproduction. Researches done on young boars show that boars‘ immunocastration with Improvac product significantly diminishes the amount of testosterone concentration in boars blood serum after the second vaccine injection (Dunshea et al., 2001; Zamaratskaia et al., 2008; Albrecht et al., 2012).
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In the world there are quite a lot of tests done on how the frequency of taking semen influences the semen quantity indexes. It was stated that if semen is taken every day spermatogenesis becomes faster, bigger number of spermatozoa in a day are secreted, but the total amount of spermatozoa and ejaculate volume are decreasing (Sernienė, 2000). Also it is very important that semen from mature boars is taken in settled rhythm as formed conditioned reflex and it influences not so much the quality of semen, but more boars jump on jackstraw, start and the duration of ejaculation, i.e. semen collection time. Before our current research all boars were used as semen donors in commercial semen production com-pany and semen was used for fertilization of females. For not to disorga-nize the accustomed boars growth and semen collection regimen, in prepa-ration as well as in all the current research period semen was collected once per week, i.e. in usual rhythm and by the same company worker.
As already mentioned, sexual functions of male and its libido are dependent very much on testosterone amount produced and circulating in animals organism blood (Ren et al., 2009). In our current research it was stated a strong correlative connection between the testosterone concentra-tion in animals blood and animals sexual activity (r=0.565; P≤0.001). Animals’ libido in preparation period was evaluated as optimal in 8±0 points. But after 2–3 weeks after the first Improvac product injection the obvious animals’ sexual attraction activity was noticed: bigger salivation, faster jump on jackstraw/mannequin, faster ejaculation start time and shorter ejaculation itself. It is connected with the growth of testosterone concentration in boars‘ blood serum after the first injection of Improvac product and later reduction of testosterone amount in blood serum. Re-searches done on young boars and described in literature have also shown, that after the second injection of Improvac vaccine the events of young boars jumping on each other, fighting among themselves, nuzzling of each other and poking significantly reduces, but it still stay higher than of surgically castrated boars (Albrecht et al., 2012).
For breeding boars whose semen is regularly used for sow insemi-nation, seeking to achieve good fertilisation results in a herd, animals‘ libido, function of testicles, morphology state of appendage gonads and ejaculated semen quality should be controlled regularly, because many factors that influence directly and indirectly have impact on breeders health, on spermatogenesis and semen quality. The evaluation of testicles functions firstly includes ejaculate volume, spermatozoa amount, sperm motility and morphology, i.e. finding pathological spermatozoa forms in
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ejaculate, because the results of fertilization just indirectly show testicle functions (Andersson and Makinen, 1999).
Sperm motility is a very important evaluation index of boars’ semen, as immotile sperm on in vivo conditions can‘t fertilize in ovule (Riske-vicienė and Januskauskas, 2006; Francavilla et al., 2007). But the motility of sperm not always shows the vitality of spermatozoa as even immotile sperm can be viable. That is why the final conclusion about semen sample quality is usually performed on semen quality indexes from several semen evaluation tests (Januskauskas et al., 2000; Olugbenga and Oniosova, 2005; Rodriguez-Martinez, 2006; Althouse, 1997 a, b). For this objective semen sample should be investigated using more than one research methods (Zhang et al., 1998; Januskauskas, 1999). In our current research the sperm motility was investigated subjectively and using computer analyser. For the sperm motility itself an injection with Improvac product had no influence (P≥0.05). Even if the biggest amount of motile sperma-tozoa was found in preparation period. This can be explained by the growth of pathological spermatozoa and especially of spermatozoa with twisted and immotile tales in ejaculates during the last weeks of the current research. Boars’ immunocastration with three doses of Improvac product had indirect negative impact on boars spermatozoon vitality. A strong ne-gative correlation connection was found between spermatozoa vitality and morphologically abnormal spermatozoa amount in ejaculate (r=–0.332; P=0.026). At the end of the current research, i.e. during the Period 3 the spermatozoon vitality has lowered 7.37% (P≤0.001) in average if compare with the preparation period. But in a Group C boars, that were vaccinated with three doses of Improvac product, the vitality of sperm at the end of the current research was 14.77% lower if compare with preparation period (P≤0.001), as in ejaculates of this group of boars the number of patho-logical spermatozoa was the biggest.
Sperm motility investigation by eye using light microscope is not complicated, is performed fast and doesn‘t need a lot of labour costs – that is why it is used widely in reproduction farms and in commercial semen collection stations too. But this motility check is very subjective, depends on technician‘s skills and mastership (Woelders, 1991). In these times in the world computer analysers on semen quality and motility are increa-singly used for more objective semen quality evaluation (Vyt et al., 2004). Usually these two research methods are used together and complement one another. During our current research sperm motility investigation was performed using both research methods: subjectively and objectively and
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we have stated that sperm motility on both methods was correlating positively in between (r=0.244; P≤0.01). Correlation between various semen quality evaluation methods is widely described in literature (Sö-derquist and Larson, 1985; Januskauskas and Rodriguez-Martinez, 1995; Huo et al., 2002; Agarwal et al., 2003; Vyt et al., 2004). Also we have noted that objectively defined sperm motility during all current research time was in average bigger (P≤0.05) comparing with subjectively defined sperm motility. Belgian scientist Vyt (Vyt et al., 2004) investigating sperm motility in few methods have also stated differences between subjective sperm motility defined using microscope and objective sperm motility stated by two different automatic systems. They have also noticed a big difference and low correlation (r<0.3) between motility percentage set by two different researchers. Authors just prove the opinion that definition of sperm motility is a very sensitive method, dependant from practical skills of a person performing it and they recommend to investigate sperm motility by automatic systems of semen quality evaluation and to analyse movements of each spermatozoon (speed, amplitude etc.) in details, what is simply impossible to see by human eye.
Immunological castration of mature boars with Improvac product was significant for reduced ejaculate volume (P≤0.001) and for spermatozoa concentration in one millilitre of ejaculate (P≤0.001). The biggest average ejaculate volume was found from Group A boars on 7th week of the current research and the smallest average ejaculate volume was set at the end of the current research. As it is known that boars from all known domestic animals are characteristic of having the biggest volume of semen (200–500 ml) in ejaculation time (At-Taras et al., 2006; McCarthy et al., 2006; Berger et al., 2008). Volume of ejaculate depends on boar‘s vesicular appendage glands and also on the size of testicles itself. In literature it is said that even 98% of ejaculate volume consists of accessory sex glands secretions, from were 60–90% is vesicular glands secretion. Vesicular glands secretion makes semen plasma. In plasma there are a lot of proteins, carbohydrates, ferments, various salts, vitamins and minerals that are needed for spermatozoa to breath. Vesicular glands secretion dilutes semen, it changes reaction of epididymis, where spermatozoa accumulate and incubate, from acidic to alkaline, energises spermatozoa breathing processes and in this way makes conditions for spermatozoa to move freely in female genital tract (Hafez and Hafez, 2000; Knox, 2003). In our current research we have found that vaccination of mature boars had no big statistically significant effect (P≥0.05) on accessory sex glands. But we
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have noticed that boars from Group B and Group C had obviously smaller vesicular gland as well as prostate if compare with control group i.e. nonvaccinated boars (P≥0.05). Development and functions of appendage gonads are controled by androgen testosterone and during our current research after boars immunocastration with Improvac product its reduction was observed. But in scientific researches on young animals it is stated that it is better to evaluate efectivity of immunocastration according to appendage vesicular glands weight reduction and not to testicles weight reduction, as with vaccine Improvac effect weight of these glands change faster than the one of testicles (Bonneau, 2010). We could explain reduction of sperm concentration in immunocastrated boars ejaculates by degeneration proceses of seminal duct found in testicles just after the animals‘ slaughtering and that it significantly reduces not just the number of emerging spermatozoa, but also the size of testicle itself (Mamina and Zhigal‘skii, 2006).
In scientific literature volume of ejaculate is connected with the size of testicles. Size of testicles is connected with the number of Sertoli cells and with percentage of seminal duct. With scientific researches it is stated that boars with bigger testicle weight have a bigger percentage of seminal duct too, where spermatogenesis takes place. These ducts have bigger diameter in bigger testicles, if compare with boars having smaller testicles (Schnickel et al., 1984). Changes of testicle size is connected as well with gonadotropin hormones FSH and LH secretion changes and a strong im-pact is done on it performing boars immunocastration.
Appearance of pathological spermatozoa in semen is an index corre-lating with testicles pathology (Serniene et al., 2005). Mature boars’ im-munocastration with Improvac product when blocking GnRH activity and raising pathological changes in testicles had an impact on pathological spermatozoa amount increase in ejaculate (P=0.004). Total average per-centage of healthy spermatozoa from the beginning till the end of the current research that lasted 15 weeks, i.e. two incomplete spermatogenesis cycles, have reduced by 27.54%. When evaluating sperm heads‘ patho-logies the most often pier forms and narrow basement heads were ob-served. It is the defect of spermatozoa nucleus, which appears because of Sertoli cells insufficiency, spermatogenesis and formulating disorder in spermatozoa nucleus condensation. When evaluating sperm tails the most often twisted tails were observed. Sperm tails are very sensitive for big variations of ambient temperature and pH. We think that appearance of these pathologies was influenced by disorder in spermatogenesis cycle,
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degeneration of seminal duct and epididymal inflammations. Spermatozoa with simple bent tails, with proximal and distal droplets are the sperm pathologies that are found the most often. And in our current research the biggest amounts of spermatozoa with cytoplasm droplets‘ changes were observed during the research time and especially big amounts of pathological spermatozoa were found during the last weeks of the current research in Group B and C boars. The appearance of these droplets or its‘ number increase in ejaculate show degenerative changes taking place in seminal epithelium (Knox, 2003; Lin et al., 2006). Taking into account pathological and degenerative changes in testicles that were caused by immunocastration we can explain why in ejaculates of Group A boars during 11 weeks of the current research the number of healthy sper-matozoa has lowered in average just by 3.7% (P≥0.05), when in ejaculates of Group B and C boars number of healthy spermatozoa has lowered by 17.5% (P≥0.05) and 36.83% (P≤0.05) respectively.
The most accurate tests results on reproductive organs are obtained with histology tests on testicles and accessory sex glands. So, unfortuna-tely just after the animal is slaughtered we can explain one or another semen quality index and their correlation found when animal was still alive. Testicles’ size as a critical factor for semen quantity is discussed a lot in scientific publications on reproduction. But testicles may become bigger because of evolved pathological processes, such as oedema, tumour or inflammation. Some of it appear unaware, some evolve gradually and that is why at the very beginning it is not easy to identify it clinically. Unfortunately in literature there is a lack of information about the con-nection between specific pathological changes and the quality of produced semen. When investigating pathological changes and its connections with semen quality it is very important to know when these processes have started in testicles, as the process of spermatogenesis, when mature sper-matozoa is developed is quite long and lasts more than two months but during that time acute processes started in testicles already became chronicle. Seeking to evaluate the impact of immunocastration for testic-les, appendage gonads, boar taint and meat quality we were comparing testicles, accessory sex glands, boar taint and meat quality indexes of animals from research groups with the same indexes of slaughtered control group animals, i.e. nonvaccinated boars. As we expected, during our current research it was stated that testicles of nonvaccinated i.e. control group boars were significantly bigger if compare with boars that were vaccinated with Improvac product (P≤0.001). This is explained by direct
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connection between testosterone concentration and testicles. Just the average testicle weight of Group A boars, that were vaccinated two times and slaughtered four weeks after the second injection of Improvac, differed slightly from control group boars testicles’ weight (P≥0.05), as testicles of mature boars are already completely developed and functioning, that is why a longer period of time is needed for negative GnRH immunocompression impact to become evident. But testicles of boars that were vaccinated two times and slaughtered 8 weeks after the second vaccine injection and of boars vaccinated three times were obviously smaller if compare with control group boars (P≤0.001). Colum-bian scientist J. Agudelo-Trujilo with co-authors (2011) after performing research with sexually mature, 26-36 month old boars have found similar differences in testicle size between vaccinated and nonvaccinated boars.
After histology tests of testicle tissue a normal usual spermatogenesis with appropriate cells for the cycle was found in control group boars’ testicles. In testicles of boars from Group A histologicaly no sexual cycle changes were found yet. This was confirmed by quite good results on semen quality indexes received earlier. As immunocompression of Group A boars (it is time from first injection with Improvac product till boars slaughtering and testicle histology test execution) lasted no longer than two months, i.e. time as long as normal boars’ spermatogenesis cycle lasts, bigger pathological morphological sperm changes and its amount varia-tions were not found. But histology tests on testicles of Group B boars have shown that just after 12 weeks after the first vaccination boars already had the following symptoms: impaired reproductive cycle, no spermatids, just stem gametes left, gigantic multinucleated cells and protein mass accumulations in tubular centre, developing chronic inflame-mation. Sperm granuloma was found in some testicle. Less Leydig cells were in connective tissue, focal infiltrations of lymphocytic and granu-locytic cells were found. This infiltration of lymphocytes and the way of violation in seminiferous tubules is based on opinion that during the inflammation an activation of autoreactive T cells is taking place (Schuppe and Meinhardt, 2005). It means that induction of testicles inflammation is connected with cells autoimmune response and that main function of immune system is disturbed. This can be the reason for functional blood and testicles barrier deficiency at the beginning of spermatogenesis, the-refore developing germinative cells can be violated and spermatogenesis itself can be weakened. Because of this reason inflammation induces spermatogenic dysfunction: the number of generated spermatozoa gets
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lower, pathological sperm forms increase, sperm motility and with that connected its fertilisation potency decrease (Tanidir et al., 2008). The third Improvac injection during the current research just made the blocking vaccine impact to GnRH stronger and after that in all testicles of boars from current research Group C chronic interstitial lymphocytic inflamma-tion was found or even clear symptoms of cells‘ degenerainflamma-tion were observed. Degenerative processes of seminal duct greatly reduces the total number of developing spermatozoa and increases the number of patholo-gical spermatozoa (Mamina and Zhigal‘skii, 2006). In the case of severe degeneration the amount of germinative epithelium itself decreases and finally spermatogenesis stops at all (Foster, 2007). So we can conclude that boars’ immunocastration had an impact for pathological processes to appear in testicles. A negative degeneration impact for testicles we have found in evaluating semen quality described above: bigger amount of pathological spermatozoa was found, concentration of spermatozoa has lowered, sperm vitality and motility have lowered too. Big changes in testicular tissues and spermatozoa morphology because of immunocastra-tion effect were also defined by Einarsson and Kauffold (Einarsson et al., 2009; Kauffold et al., 2010; Einarsson et al., 2011) in research with young animals. Agudelo-Trujilo (2011) have detected reduced spermatogenesis cycle and Leydig cells number in boars group vaccinated against GnHR, if compare with nonvaccinated boars.
In literature the data is given that animal species, sex, age, physio-logical condition, stress, also ration, living conditions and even season have influence for biochemical blood parameters (Máchal et al., 2007; Kahn and Line, 2010; Chmielowiec-Korzeniowska et al., 2012). But we haven‘t found data that boars vaccination against GnHR has influence on biochemical blood parameters. Also in our current research we have stated that immunological castration with Improvac product for mature, sexually adult and already in use for reproduction boars blood biochemical para-meters had no statistically significant effect (P≥0.05). Like in other scien-tists’ works, average meanings of biochemical blood indexes in prepara-tion period, as well as in research period were in the boundaries of phy-siological norm (Rekiel et al., 2008; Nerbas, 2008).
Main chemicals responsible for unpleasant boar taint are androste-none, skatole and indole (Garcia-Regueiro and Diaz, 1989; Moss et al., 1993; Annor-Frempong et al., 1997; Dijksterhuis et al., 2000; Rius and Garcia-Regueiro, 2001; Zamaratskaia, 2004, Haugen et al., 2012). In our current research to find immunocastration impact on mature animals boars
