Lucerne, Switzerland

18–19 October 2018

29th EuropeanAI VETS-Meeting

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The Local Organizing Committee is proud to welcome you to the 29th AI-VETS Meeting in Lucerne, Switzerland. The meeting will take place on Oct 18th – 19th 2018. Additionally, touristic activities are planned for the afternoon of Friday, Oct 19th and excursions to the facilities of Swissgenetics and SUISAG will be offered on Saturday Oct 20th.

Lucerne is located in the heart of Switzerland and one of the most beautiful cities, located on the shore of the «Vierwaldstättersee» not far from the «Rütli», the place, where Switzerland has been founded in 1291.

Venue of the conference will be the Hotel Astoria, located in the center of Lucerne.

The annual AIVETS Meeting is the exchange platform for more than 100 specialists working in in the field of artificial insemination in livestock. The conference is organized under the supervision of the RepVet group, an advisory group of EFFAB and is hosted every year in a different European country.

The Local Organizing Committee

Welcome address

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RepVet BoardErwin Hasenpusch (ADT, Chair)

Friedrich Führer ((ZAR, Treasurer)

Sabine Brüning ( ADT, Secretary)

Eveline Willems (Topigs Norsvin)

Olivier Gérard (ALLICE)

Scientific program groupJaap Bosch (coordinator)

Marleen Broekhuijse

Sabine Brüning

Dimitri de Meyer

Friedrich Führer

Eveline Willems

Ulrich Witschi

Local organizing committeeManuela Falk (Swissgenetics)

Christin Oehler (SUISAG)

Ulrich Witschi (Swissgenetics, Chair)

Sandra Woodtli (Swissgenetics)

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Congress venueHotel ASTORIAPilatusstrasse 296002 Luzernwww.astoria-luzern.ch

For car parking: please use the public parking spaces / garages.

General Information

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AIVETS-Dinner

*** Don’t forget to admire the pleasant view while you are on the bridge ***

• • • • • • • Direction from Hotel ASTORIA (approx. 1 km – 15 minutes)

At Hotel ASTORIA hand left and head down to Pilatusstrasse (approx. 20 m)On Pilatusstrasse go right and follow the street until you arrive at main station (about 500 m)Once arrived in front of the main station turn left and you will see a large bridgeGo over the bridge (Seebrücke) and follow the right lakeside until you arrive at the restaurant ship WILHELM TELL (about 600 m)

AIVETS-Dinner

October 18th, 2018 – 20h00Schiff-Restaurant WILHELM TELLLandungsbrücke 9, 6006 Luzern

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Touristic Activity

City Tour

October 19th, 2018, 14:00–16:00, Meeting point: Lobby Hotel ASTORIA, Your guide: Heidi Muffler

Excursion to Swissgenetics

08:30 Departure

Meeting point: in front of the hotel ASTORIA

09:00 Langnau

11:00 Mülligen

12:00 Lunch

13:00 Departure

14:00 Drop-off at Zurich Airport

Excursions Swissgenetics / SUISAG

October 20th, 2018

Excursion to SUISAG

10:00 Departure

Meeting point: in front of the hotel ASTORIA

10:30 Semen Center Knutwil

11:30 Lunch

12:30 Departure

14:00 Drop-off at Zurich Airport

Thursday, Oct 18th, 2018

08:30-12:30 Registration (Hotel Astoria, Level 7, Hall)

08:30-10:30 RepVet meeting (Room 2)

10:30-12:00 Pig (Room 3) and Cattle (Room 2) QualiVet meetings (parallel)

12:00 Lunch (Penthouse)

13.00-13.15 Welcome to Switzerland (Room 2)

13:15-15:30 Plenary session I: Future Challenges for the AI – Industry (Room 2)

16:00-17:00 Cattle session I: Sire care (Room 2)

Pig session I: Boar health (Room 3)

17:00-18:00 Sponsors and exhibitors presentations (Room 2)

18:00-18:30 Reports from RepVet and Pig/Cattle QualiVet (Room 2)

20:00 AI-Vets Dinner

Conference Program

Friday, Oct 19th, 2018

08:00-10:45 Registration (Hotel Astoria, Level 7, Hall)

08:30-10:30 Plenary session II: Changes in Regulations and advances in technology (Room 2)

11:00-12:30 Cattle session II: Semen Processing and Customer solutions (Room 2)

Pig session II: New technologies: how do they fit in routine semen processing (Room 3)

12:30-12:45 Closing ceremony (Room 2)

13:00-14:00 Lunch (Penthouse)

14:00-18:00 Social programme (touristic activity)

Open evening

Saturday, Oct 20th, 2018

08:30-14:00 Excursion to Swissgenetics Rearing Station and AI-Centre (In front of the Hotel Astoria)

10:00-14:00 Excursion to SUISAG (In front of the Hotel Astoria)

Approx. 14:00 Drop-Off at the Airport in Zürich

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Scientific Programme

Thursday Oct 18th 2018

time topic Speaker

13:00–13:30 Welcome to Switzerland Ulrich Witschi

13:30–15:30 Plenary Session I: Future Challenges for the AI – Industry (Room 2)Chair: Maarten Moleman

13:30–14:00 Genomic detection of inbreeding depression and heterosis for semen quality traits in AI bulls

Johann Sölkner

14:00–14:30 Exploiting genomic data to improve male fertility Hubert Pausch

14:30–15:00 Coffee Break

15:00–15:30 Gene editing methods in food animals – another breeding tool for genetic improvement

Tad Sonstegard

15:30–16:00 Effects of exogenous factors on onset of puberty in bull calves Heiner Bollwein

16:00–17:00 Cattle Session I: Sire care (Room 2)Chair: Frank Bosselmann

16:00–16:30 Opportunities to enhance claw health by nutrition and genetic selection

Menno Holzhauer

16:30–17:00 The effectiveness of air pressure systems as a means of preventing the presence of Culicoides midges in bull barns.

Jaap Bosch

16:00–17:00 Pig Session I: Boar health (Room 3)Chair: Christin Oehler

16:00–16:30 Boar reproductive ultrasound examination predicting future sperm quality?

Svenja Beyer

16:30–17:00 Do’s and don’ts in boar feeding Robert Tabeling

17:00–18:00 Sponsors presentations (Room 2)Chair: Sandra Woodtli

18:00–18:30 Reports from RepVet / QualiVet (Room 2)

20:00 AIVETS-Dinner

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Friday Oct 19th 2018

time topic Speaker

08:30–10:30 Plenary Session II: advances in technology (Room 2)Chair: Marleen Broekhuijse

08:30–09:00 The risk of a Mycoplasma bovis infection in a dairy herd by semen processed according to the EU standards

Vera Haapala

09:00–09:30 (Micro)plastics: The path from products to environmental distribution

Denise Mitrano

09:30–10:00 The continuous need for antibiotic stewardship in animal production and the response to a changing environment

David Speksnijder

10:00–10:30 Coffee Break

10:30–11:00 Optimal Screening Tests of Semen Quality in early pubertal bulls destined for Artificial Insemination

Ciara O’Meara

11:00–12:30 Cattle session II: semen processing (Room 2)Chair: Attila Szakács

11:00–11:30 M. bovis: PCR screening of the semen production Karina Elkjaer

11:30–12:00 Quality control: the multicolor assay and its biological relevance Eleni Malama

12:00–12:30 Semen quality and field fertility of peripubertal bull ejaculates Marleen Broekhuijse

11:00–12:30 Pig Session II: New technologies: how do they fit in routine semen processing (Room 3)Chair: Dimitri de Meyer

11:00–11:30 Impact of transport stress on boar semen quality during long-term storage

Martin Schulze

11:30–12:00 Modern technics to improve semen quality and laboratory management

Sabine Brüning

12:00–12:30 Reducing antibiotics in boar semen extenders Christin Oehler

12:30–12:45 Closing ceremony (Room 2) LOC, Chair RepVet

14:00–18:00 Social programme (touristic activity)

Saturday Oct 20th 2018

08:30 Excursion to Swissgenetics AI-Centre and Rearing Station (in front of the hotel)

10:00 Excursion to SUISAG (in front of the hotel)

Ca. 14:00 Drop off at Zurich Airport

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Plenary Session I: Future challenges for the AI-Industry

Genomic detection of inbreeding depression and heterosis for semen quality traits in AI bullsJohann Sölkner 1, johann.soelkner@boku.ac.atNegar Khayatzadeh 1, Maja Ferenčaković 2, Miroslav Kapš 2, Gabor Mészáros 1, Ino Curik2

1 University of Natural Resources and Life Sciences (BOKU) Vienna2 University of Zagreb

The availability of high throughput genomic data has revolu-tionized cattle breeding. In big populations, young bulls are now selected based on their genomic profiles based on 50,000 single nucleotide polymorphism (SNP) markers. These ge-nome-wide SNP markers may be used to learn much more. Amongst others, the genetic mechanisms governing inbreed-ing depression and heterosis may be detected. In two studies, we explored which regions of the genome are responsible for inbreeding depression for sperm quality in Austrian Fleckvieh bulls and for the effects of heterosis, also for sperm quality, in Swiss Fleckvieh bulls.

The level of inbreeding can be assessed very accurately from SNP data. If long segments of consecutive SNP in a region are homozygous for an animal, the only valid explanation is that this animal received identical segments from both parents, de-rived from a common ancestor. The proportion of the genome in such ‘runs of homozygosity’ (ROH) reflects the inbreeding coefficient of an animal. In this study, we assigned a code to each SNP of an animal, being 0 if this SNP was not in a ROH and 1 if it was in a ROH. A total of 19,720 ejaculates of 554 bulls were analysed for ~41,000 SNP, considering also the additive genetic value of each SNP as well as many environmental fac-tors. We found several regions significantly associated with status of inbreeding and searched for genes in these regions. For total number of sperm, Sperm Flagellar Energy Carrier Pro-tein (SLC25A31) on chromosome 17 was among the candi-dates. For percent of alive sperm, Spermatogenesis Associated 16 (SPATA16) gene on chromosome 1 gave the most significant signal. Another significant signal was found in the vicinity of Spermatogenesis Associated 18 (SPATA18) gene, which en-codes a so-called “mitochondrion-eating protein”.

Crossbreeding is a technique that is frequently used in animal breeding. In pigs and poultry, terminal crosses are used. In cat-tle, composite (often called ‘synthetic’) breeds are more fre-quent. Swiss Fleck vieh (SWF) is a composite of Simmental (SIM) and Red Holstein Friesian (RHF), with pedigree propor-tions of 12.5 to 87.5 % of either breed. The development of the composite started around 1970, so that current Swiss Fleckvieh animals are the product of up to 10 generations of crossbreed-ing. As it is simple to find whether a SNP is inbred or not, it is also comparatively simple to find whether the two alleles of a SNP in a composite animal are both derived from one breed (code 0 in our analysis) or they come from two different breeds (code 1). The analysis included 41,749 ejaculates for 1169 SWF, SIM and RHF bulls, and 38,205 SNP. Percent of alive sperm was the trait investigated in this study. In the genome wide-analy-sis, we found a heterosis effect of 1.6% alive sperm, indicating strong heterosis, as expected for fertility traits. As for breed proportion, pure RHF showed an advantage of 0.6% over SIM. Several genomic regions were potentially causative for hetero-sis in the trait. PKDREJ is a protein coding gene on chromosome 5 which encodes a member of the polycystin protein family. This protein plays a role in human reproduction and fertiliza-tion. THEG gene is a protein coding gene on chromosome 7 and is expressed in a nucleus of haploid male germ cells possibly involved in spermatogenesis. SPAG4 is a protein coding gene on chromosome 13, involved in spermatogenesis and mainte-nance of the general polarity of the sperm head.

In conclusion, the search for genes involved in inbreeding de-pression and heterosis for bovine sperm quality traits yielded very promising candidate genes. The analyses of bovine sperm are elucidating for better understanding of the genetic mecha-nisms linked to male fertility.

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Impaired male fertility has been described in many species in-cluding humans. Although more than 15% of couples world-wide suffer from impaired fertility and half of these cases are attributable to “male factor” sub- or infertility, genetic mecha-nisms underlying male reproductive performance are poorly understood. Because the heritability of male fertility is low, large cohorts of men with both genotypes and phenotypes are required to identify genomic loci controlling reproductive per-formance. Large genotyping endeavors are underway (e.g., the UK Biobank) in order to collect dense genotypes for hundreds of thousands of humans. However, phenotypes for male repro-ductive performance are not readily available for these cohorts and collecting relevant fertility data is difficult in humans.

This situation is different in livestock populations: millions of bulls and boars have been genotyped using dense microarrays in order to estimate their genomic breeding values. Moreover, highly specialized artificial insemination (AI) centers regularly collect ejaculates from tens of thousands of bulls and boars under controlled environmental conditions. To monitor semen quality, all ejaculates are closely examined immediately after collection and only ejaculates that comply with the current rec-ommendations for AI are retained. Moreover, (ejaculate-specif-ic) non-return rates are recorded in order to monitor male fer-tility and insemination success. The availability of dense genotypes, fertility records and semen quality data makes livestock populations highly amenable to the genetic mapping of genomic loci that underpin male reproductive performance, thus allowing for fundamental insights into biological mecha-nisms underlying mammalian reproduction. The analysis of more than 15 million artificial inseminations of 7’962 Fleckvieh bulls that have been genotyped at 650’000 SNPs enabled us to identify two haplotypes located at bovine chromosomes (BTA) 5 and 19 that are associated with very low insemination suc-cess. It turned out that the associated haplotype on BTA19 car-

ries a nonsense mutation in a gene encoding a sperm trans-membrane protein that is likely required for sperm-egg interactions in vivo. At the BTA5 region associated with poor reproductive performance, we identified a mutation in a gene that encodes a protein that is involved in inducing acrosome reaction in vivo. We are currently investigating if impaired acrosome reaction prevents fertilization of sperm from bulls that are homozygous for this mutation. Because bulls that are homozygous at those variants produce semen with normal sperm concentration, viability and (progressive) motility, their semen was used for more than 45’000 inseminations, but none of them was successful. In the meantime, we developed and implemented direct gene tests on custom genotyping microar-rays in order to identify infertile bulls before their semen is used for artificial inseminations. Impaired fertility in spite of normal semen quality (i.e., idiopathic sub-/infertility) is a major threat particularly to genomic breeding programs because per-tinently affected bulls may have been used for thousands of artificial inseminations before low insemination success be-comes apparent.

However, apart from idiopathic fertility disorders, artificial in-semination centers sometimes report bulls and boars with poor semen quality. We identified causal mutations for disorders that manifest multiple aberrations of the sperm flagellum or asthenospermia in Finish Ayrshire cattle and we are currently analysing an acrosome defect in Yorkshire boars that prevents fertilization in vivo.

Moreover, bulls and boars that are kept at artificial insemina-tion centers are an ideal cohort for association testing between dense markers and semen quality data. Results of GWAS with semen quality parameters as quantitative traits will be present-ed as well as their implications for genomic approaches to-wards improving male fertility will be discussed.

Exploiting genomic data to improve male fertilityHubert PauschETH ZürichAnimal GenomicsEschikon 27 | EHB 218315 Lindauhubert.pausch@usys.ethz.ch

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Gene editing methods in food animals – another breeding tool for genetic improvementTad S. Sonstegard* and Daniel F. CarlsonAcceligen – Recombinetics Inc., St. Paul, MN, USA* corresponding author – tad@recombinetics.com

Gene editing based on site-directed nucleases is recognized as a breeding method best suited to introduce high effect animal health, well-being, production, and disease resistance alleles into naïve populations of food animals. These methods provide new opportunities in the marketplace with great potential to help meet global food security challenges related to animal protein production. However, there has been only a few pri-vately funded initiatives attempting to bring edited animals to market; suggesting commercial providers of elite genetics are

still reticent to apply this technology as a method for animal improvement. The pre-commercial deployment of animal wel-fare traits for genetic improvement in food animals and poten-tial economic value to producers will be reviewed to provide background on the lack of biosafety risks associated with this technology. Beyond science-based risk, other important factors affecting future acceptance and widespread adoption of gene editing technology as a primary tool of animal breeding will be discussed.

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Early sexual development of bulls has always been important for cattle breeders because they want to reduce production costs and shorten the generation intervals to increase genetic gains. However, because of the introduction of genomic selec-tion in cattle breeding a few years ago, the relevance of the reproductive performance of bulls has increased tremendously. Using this new method, the breeders are able to obtain infor-mation about the genetic value of the bulls at either the embry-onic period or immediately after birth. With this information, they may be able to obtain semen from bulls with high genetic value earlier. One factor that limits this goal of the breeders is the high variability in the onset of puberty and sexual matura-tion in bull calves. For example, bulls from European breeds reach puberty between 34 and 54 wk after birth and the time to sexual maturation ranges from 42 to 66 wk of age.

Since more than three decades several attempts have been per-formed to decrease the age at puberty by hormonal treat-ments, especially by repeated injections of GnRH in calves be-tween 4 and 8 weeks of age. By using such strategies, the age at the time of puberty could be decreased by about 4 to 6 weeks. However, these treatments are time consuming and not well accepted by the consumer.

Therefore, in the last decades a series of attempts have been made to improve reproductive performance of bulls via opti-mizing nutrition. Although an increase in energy uptake during the post-weaning period of calves led to a faster growing rate, it had no positive effects on sexual development. In contrast, a high-nutrition diet during the infantile and prepubertal periods reduced the age at puberty of the bulls and increased the size/weight of the testis and the epididymal sperm reserves. This

faster sexual development was associated with an increased transient LH peak, which seemed to be mediated by an increase in serum IGF-I concentrations.

It has to be emphasized that sexual development of bull calves depends not only on nutrition but also on the health of the calves. Calves with a history of pneumonia grow significantly less than healthy calves, and the effects of a higher level of a high nutrition level immediately after parturition on sexual de-velopment is totally abolished in those calves.

Interestingly, the development of bull calves is affected by their nutrition not only after but also before birth. In contrast to the positive effects of high feed intake by the bull calves during the first weeks after birth, a high-nutrition diet fed to the mother during the first trimester seems to have negative effects on the development and the reproductive performance of their off-spring later in life. It has not yet been determined whether there is also an effect of periconceptional feed intake by the cows on the development of their male offspring, as has been reported in sheep.

Several studies have demonstrated that modifications in feed intake have an influence on the hypotha¬lamic–pituitary–go-nadal axis, which might be mediated by serum IGF-I con-cen¬trations, but the exact mechanisms responsible for the interaction between nutrition and the subsequent develop-ment of offspring are not yet clear. Therefore, further studies are necessary to obtain a better understanding of the phenom-enon of effects of nutrition on sexual development in bulls to be able to optimize the performance of young bulls.

Effects of exogenous factors on onset of puberty in bull calves H. Bollwein*, F. Janett, M. KaskeClinic of Reproductive Medicine, Vetsuisse-Faculty, University of Zurich* Corresponding author:

Heinrich Bollwein, Clinic of Reproductive Medicine, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich Phone: 0041-44-63 58242 hbollwein@vetclinics.uzh.ch

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Cattle Session I: Sire care

Opportunities to enhance claw health by nutrition and genetic selectionMenno Holzhauer, DVM, PhD, Dip. ECBHMGD Animal Health, P.O. Box 7, 7400 AA Deventer, The Netherlandse-mail: m.holzhauer@gdanimalhealth.com

Claw disorders and lameness are besides mastitis and fertility a very important and costly health issue in dairy cattle. Thereby claw-disorders are mostly long lasting and related to serious discomfort and pain. This makes it an important welfare issue also. Both management, by improved housing and nutrition, and genetic selection are tools to improve leg and claw health.

Nutritional components for good claw-horn quality are among others amino and fatty acids, minerals (calcium, zinc, manga-nese etc.) and vitamins (among others biotin) and optimal sup-ply results in good horn and skin quality and less susceptible for infectious and non-infectious disorders. Since the beginning of this year GD provides a validated bulk-milk monitoring for some main components (Zn, Mn and biotin) and the results seems to be promising for improvement. Monitoring by bulk milk is simple where the product is easy accessible and pro-vides information of the final result of intakes and ruminal con-sumption and production of for example biotin. Monitoring nutritional components is of importance for the animals pres-ent at this moment and the next future in the herd and optimal supply must result in good horn quality, less lame cows and optimal farm results and unburdening of the livestock farmers.

Genetic selection improvement provides more permanent gains (next generation), contributing to a better durability. Several different sources of claw-health data like claw trimmer

data and veterinary diagnosis are available. Especially informa-tion by claw trimmers during regular «preventive» claw trim-ming are widely available and showing most reliable and most promise. Many observations are needed to produce genetic evaluations with high reliability of most claw-health traits, so the use of auxiliary traits that may have positive genetic corre-lations with direct measures of claw health may be necessary. It is important to establish recording systems that use common trait definitions and recording standards to ensure that evalua-tions are based on high-quality data/information. Incentives, such as an easy-to-use electronic recording system will help to motivate claw trimmers to participate in data collection ef-forts. Another motivator might be good feedback of the profits of this information to farmers and herd advisors. Claw-health traits must of course be added to the total merit indices with sufficient economic weight to enable genetic improvement. Reference populations for genomic evaluation may need to in-clude cows due to lack of bulls with high-reliability traditional evaluations.

To conclude: a combination of monitoring good vitamin and mineral supply (short term) and genetic improvement by selec-tion of sires with good genetic claw health figures (long term) will result in better welfare and durability.

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Following the bluetongue virus epidemic in 2006-2008 in the Netherlands, CRV has equipped the production barns with an air pressure system at their production locations, with the in-coming air blown through perforated tubes and leaving the building over the housed bulls along the sides of the building. The idea is that the continuous, filtered, flow of air in the barn will reduce – and maybe totally prevent – the inflow of Culi-coides midges.

A study was performed in 2006 to get an impression of the ef-fectiveness of air pressure systems as a means of preventing the presence of Culicoides midges. Two barns, one with and one without the air pressure system were compared with re-spect to the number of midges caught outside the barns. The study showed a reduction in midges in the barn with the air pressure system. However, the study was carried out in the au-tumn with relatively low number of midges outside so that no clear conclusions could be drawn.

In daily practice it was difficult to get a good climate in the barns at hot summer days.

In 2016 and 2017 the production barns 1 and 2 in Giekerk and the production barn in Harfsen were reconstructed with bigger boxes per bull. The number of bulls was reduced per barn. Fur-thermore, the air pressure systems were renewed, resulting in higher ventilation capacity, bigger air tubes and a better air circulation in the barns. The quarantine barns of both locations were not provided with the air pressure system, yet.

A new study was carried out in the summer of 2017, during a period in which the abundance of midges in the air was pre-sumed to be high. For three consecutive nights, midges were trapped in a production barn (with air pressure system), in a quarantine barn (without air-pressure system) and outside the buildings at both locations using a standard Onderstepoort Veterinary Institute (OVI) suction light trap. The study was car-ried out by Wageningen Bioveterinary Research in Lelystad.

Results Most Culicoides midges were trapped outside the buildings while in the production barns only very low numbers of midges (0 – 3 midges /barn/24 hours) were trapped. The differences between trapped midges in the quarantine barns and outside ranged widely by day and location. In the quarantine barn of the production barns 1 and 2 in Giekerk large numbers of midg-es were trapped (comparable with the number of midges

trapped outside and sometimes even more), while in the quar-antine barn in Harfsen considerably less midges were trapped compared to outside the buildings, but about twice as much as in the production barn. At both locations, the number of midg-es trapped in the quarantine barns were higher than in the production barns but less than outside.

The effectiveness of air pressure systems as a means of preventing the presence of Culicoides midges in bull barns.

Authors: Dr. J.C. Bosch, veterinarian; Dr. Ir. A.R.W. Elbers, Wageningen Bioveterinary Research Lelystad.

Figure 1. Total number of Culicoides midges trapped with an OVI suction light trap by day in the production barns, quarantine (Q) barns and outside the buildings of the CRV locations Giekerk + Harfsen.

Conclusions There is a clear preventive effect of the air pressure system on the presence of Culicoides midges in the production barns. However, complete absence of Culicoides midges in the pro-duction barns cannot be guaranteed by only the air pressure system. This might probably be due to the still relatively open structure of the barns (for example no air-locks). Supplementa-ry treatment of the bulls with deltamethrin or permethrine is therefore still advised.

On the location in Giekerk, a higher number of midges inside than outside the quarantine barn in Giekerk was observed. It is speculated that this might be caused by the fact that the gen-eral weather conditions in Giekerk with more wind, stimulate midges to seek a more comfortable environment in the barn.

e-mail: jaap.bosch@kpnmail.nl armin.elbers@wur.nl

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Pig Session I: Boar health

Boar reproductive ultrasound examination predicting future sperm quality?S. Beyer 1, F. Beyer 1, M. Kleve-Feld 2, R. Bortfeldt 1, M. Jung 1, M. Schulze 1*1 Institute for the Reproduction of Farm Animals Schönow, Bernau, Germany2 Pig Improvement Company, Hendersonville, TN, USA* Corresponding author.

Tel.: +49 3338-709822; Fax: +49 3338-709810 E-mail address: m.schulze@ifn-schoenow.de (M. Schulze)

New ways to predict fertilizing performance in young artificial insemination (AI) boars are very important for breeding com-panies to make their selection process more efficient. Due to the large impact boars have on sow herd productivity, it is vital to select them based on their reproductive capability and car-cass value. The evaluation of a boars´ eligibility for use in AI should include the review of its´ breeding history, a physical examination (external genitalia and body conformation) and a semen evaluation. Puberty of AI boars starts around 150 days of age by finishing the first spermatogenesis cycle. Breeding maturity is reached at 7 to 8 months of age. During sexual de-velopment, the highest increase of testicular volume occurs between 100 and 180 days of age. Today, B-mode ultrasound is a proven and noninvasive method to have a detailed look at the organs macrostructure. For the assessment of microstructures in vivo, the Grey-scale analysis (GSA) of ultrasound images seems to be an interesting tool, which offers different parame-ters providing information about the pixel quality and quantity. Recent studies have shown that GSA is a useful method to pre-dict future sperm quality in dogs and rams (England et al. 2012; Ahmadi et al. 2012). The objective of the current study is to characterize the testicular development and morphological changes of 1.500 Piétrain boars (Line 408, Pig Improvement Company) for commercial use in AI at defined ages throughout pubertal development using GSA. All male piglets are weaned at 21 days of age, maintained in a temperature-controlled facil-ity with ad libidum water availability and standard feed diets formulated to meet PIC requirements. Prospect breeding boars are tested for average daily gain (ADG) between 100 (+/-5) and 170 days (+/-10) of age. By the end of the test period, body weight, backfat and muscle depth are measured; followed by

leg scoring and a general visual inspection. All collected pa-rameters are uploaded to a database system (PICtraq®) where pedigree, litter and genome information are stored. The sum of traits along with additional parameters is used to calculate the overall breeding index of the animals. Scrotum, testes and epididymis will be estimated at day 100 and 170 with a stand-ardized procedure using B-mode ultrasonography with Logiq e R7® (GE healthcare) and a 5-13-mHz dual frequency 12L-RS linear transducer. GSA will be performed on a freeze image. The mean, minimum and maximum grey values and heteroge-neity are determined for each region of interest using the Im-age pro premier® (Media cybernetics) software package. Cus-tomized software is used to define the mean gradient value, mean contrast and normalized grey-scale histogram width at 5% mark. At each age, testicular volume will be calculated us-ing a diameter estimated by ultrasonography according to Wicke et al. (1991). In order to evaluate the boar`s performance and sperm productivity in comparison to the GSA analysis, ejaculate volume, sperm concentration, total sperm number, percentage of morphologically intact spermatozoa, progres-sive motility and thermo-resistance will be determined using composite data (average values) obtained from the first five semen collections. Boar libido will be scored based on a stand-ard that was characterized by recording: the time boars en-tered to first mount (1), the time taken to start ejaculation (2) and the duration of ejaculation (3). All data will be collected during 2018/2019 and corrected for possible seasonal effects on semen and testis size. Our long-term goal is to improve the selection process of young AI boars. Therefore, a high stand-ardization of the analyses and the use of the latest technology are the key to the success.

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The feeding of boars used for artificial insemination (AI-boars) is a very specialized field in boar management due to its very limited size and recognition) within pig production worldwide. Only little work was done in this field in recent years, since the feed for boars is not a significant economical factor compared to its impact on the reproduction of swine. Semen production is primarily focused on breeding, semen processing, hygiene, preservation, transport and storage.

Aspects of boar feeding crucial for performance in boar studs are: • Maintenance requirements• Fertilizing potential (capability) of semen • Soundness of limbs • Dietary problems

Maintenance requirements With the beginning of semen production at 7-9 months of age young AI-boars are still in a growing stage which is estimated to be 400 g/d up to a weight of 180 kg bodyweight. For the longevity it is crucial to control the weight gain (in older boars 200 g/d).

Table 1: Nutrient requirements for adult AI boars*

weight (kg) 120-180 180-300

young boar adult boar

age month 7 10/11 -…

weight gain (kg/d) 400 0-200

ME MJ 30 30

crude protein g 450-510 450-510

calcium g 15 15

phosphorus g 12 12

sodium g 3 3

lysine g 25 25

metionine+cystine g 17 17

* Supplemente zur Tierernährung, Kamphues et. al 2014

Influence on reproductive performanceBoar libido is affected by nutrition especially with low protein diets/intake and over-conditioning by excessive weight gain

which leads to fatter and lethargic animals. Too little feed and energy intake results in a reduced semen volume and sperm production. Due to the fact that sperm production and the pro-cess to mature semen lasts appr. 48 days, all changes in nutri-tion have to be assessed after this period.

The use of essential amino acids was object of several studies in the past with the result that there are specific requirements for the AI-boar for lysine, methionine and cysteine, but that the required amounts are not as high as expected.

For vitamin E and selenium supplementation there are some strategies to improve the antioxidant properties in semen which supports the semen maturation and therefore the semen quality. Furthermore, vitamin C is added in some areas to re-duce summer heat stress.

Table 2: Dietary nutrient specifications for AI boars* (2.5–3 kg/d)

nutrient unit/kg feed

ME MJ 11-12

crude protein g 180

crude fiber g 70

calcium g 6.0

phosphorus g 4.5

sodium g 1-1.5

lysine g 10-12

metionine cystine g 8-9

selenium mg 0.15-0.2

vitamin A IE 4000

vitamin D IE 240

vitamin E IE 40

vitamin B 2 mg 3.2

vitamin B 6 mg 1

vitamin B 12 mg 0.015

cholin mg 1200

* Supplemente zur Tierernährung, Kamphues et. al 2014, Empfehlungen zur Energie- und Nährstoffversorgung von Schweinen, GfE 2006

Do’s and don’ts in boar feedingCorresponding author:Dr. Robert TabelingHead of technical service and market access management swine MSD Animal Health Intervet Deutschland GmbHrobert.tabeling@msd.deTel: + 49 89 45614 – 282 Mobil: + 49 172 8347531

In the group of mycotoxins esp. zearalenone in feed can affect semen production by reducing semen output, fertilization abil-ity due to negative effects on sperm motility and acrosome in-tegrity. Beyond that aflatoxin and vomitoxin have detrimental effects on semen quality. Therefore, the use of binding agents is discussed widely but much more important is the control of ingredients.

• Soundness of limbs Besides breeding aspects and semen production musculoskele-tal problems are the most important issues for boar culling. Valuable boars can often not be kept for an ideal period be-cause of leg weakness or claw problems. The correct minerali-zation and reduced weight gain are crucial in this complex, yet

another problem is that the meaning of claws quality is often underestimated. In combination with the housing conditions supplementations of Biotin, Zn, Cu, Mn are nowadays dis-cussed to improve the integrity of skin and claws.

• Dietary problemsIn the rare case of digestive tract disturbances feed hygiene is often the cause. Especially water quality (water resting in the water pipes for too long), feed storage/administration, clean-ing of troughs/silos and intake of unsuitable bedding material can cause the oral uptake of bacteria. Intestinal infections and/or endotoxin production can lead to serious diseases and affect sperm quality as well.

Your professional partner in pig production

SUISAG | Allmend 8 | CH-6204 Sempach | Tel. ++41 41 462 65 50 | www.suisag.ch

Artificial insemination

Health service

GeneticsGenetics

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Plenary Session II: Advances in technology

BackgroundMycoplasma bovis has recently emerged in new countries and in addition to welfare effects, it causes economical losses to cattle industry. M. bovis causes a variety of signs, most com-mon being respiratory disease, mastitis and joint infections. M. bovis is resistant to many antibiotics, making it difficult to treat.

The main source of M. bovis infection is animal contact. Exam-ples of other possible, but less common sources, include con-taminated equipment and environment, airborne transmission, other animal species, artificial insemination (AI) and embryo transfer.

M. bovis was detected for the first time in Finland at the end of 2012. During 2012−2015, altogether 20 Finnish dairy farms were infected with M. bovis, which represented 0.26% of all 7 600 Finnish dairy farms in 2015.

Case descriptionTwo closed dairy farms with good biosecurity were infected with M. bovis in Finland in 2015. These farms were investigated in more detail as the epidemiological data did not suggest a typical infection source. M. bovis was detected from routine clinical mastitis milk samples (PCR) from both farms. Both cows had been inseminated with the same bull four weeks be-fore the clinical signs. The semen of this bull collected from the other farm and some other lots of the same bull from the breed-ing company were M. bovis positive. Other bulls used to insem-inate the M. bovis mastitis cows in these farms were negative. In addition, cgMLST supported the epidemiological results. Isolates from the two farms clustered separately from the other Finnish isolates, although limited diversity among the Finnish isolates was seen. These results support the finding that M. bovis positive semen used in insemination was the source of the M. bovis infection in these two dairy farms.

Even though infection via semen appears to be a rare, semen should be considered as a possible source of infection. This should be noted especially in areas free of M. bovis, as well as

in herds with high biosecurity. Global trade in semen might transmit the disease to a new country. As Mycoplasma bovis is intrinsically resistant to some antibiotics and develops resist-ance to new ones, the antibiotics used in semen extenders should be re-evaluated to obtain M. bovis-free semen.

References

Bielanski, A., Devenish, J., Phipps-Todd, B., 2000. Effect of Mycoplasma bovis and Mycoplasma bovigenitalium in semen on fertilization and association with in vitro produced morula and blastocyst stage embryos. Theriogenology. 53, 1213-1223.

Calcutt, M.J., Lysnyansky, I., Sachse, K., Fox, L.K., Nicholas, R.A.J., Ayling, R.D., 2018. Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements. Transbound. Emerg. Dis. 65, 91-109.

Gloria, A., Contri, A., Wegher, L., Vignola, G., Dellamaria, D., Carluccio, A., 2014. The effects of antibiotic additions to extenders on fresh and frozen-thawed bull semen. Anim. Reprod. Sci. 150, 15-23.

Haapala, V., Pohjanvirta, T., Vähänikkilä, N., Halkilahti, J., Simonen, H., Pelkonen, S., Soveri, T., Simojoki, H., Autio, T., 2018. Semen as a source of Mycoplasma bovis mastitis in dairy herds. Vet. Microbiol. 216, 60-66.

Maunsell, F.P., Woolums, A.R., Francoz, D., Rosenbusch, R.F., Step, D.L., Wilson, D.J., Janzen, E.D., 2011. Mycoplasma bovis Infections in Cattle. J. Vet. Intern. Med. 25, 772-783.

Nicholas, R.A.J., Ayling, R.D., 2003. Mycoplasma bovis: disease, diagnosis, and control. Res. Vet. Sci. 74, 105-112.

Wrathall, A.E., Ayling, R.D., Simmons, H.A., 2007. Risks of transmitting mycoplasmas by semen and embryo transfer techniques in cattle, sheep, goats and pigs. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 2, 31 pp.

The risk of a Mycoplasma bovis infection in a dairy herd by semen processed according to the EU standardsVera Haapala, DVM, Specialized in production animal medicine.University of Helsinki, Faculty of Veterinary Medicine. Finland. vera.haapala@helsinki.fi

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(Micro)plastics: The path from products to environmental distributionDr. Denise M. MitranoEawag: Swiss Federal Institute of Aquatic Science and Technology. Überlandstrasse 133, 8600 Dübendorf, SwitzerlandDenise.Mitrano@eawag.ch

Plastic is an integral part of our daily lives in modern society, ranging from high-tech medical devices to clothing to sin-gle-use packaging for food and products. Consumers are often at the nexus of plastic waste reduction: they can be encour-aged to use less plastic (for example, no bags given at grocery stores), or through effectively recycling, such as PET drink bot-tles. There may be other ways to help reduce plastic in the en-vironment, such as substitution of some traditional plastics for biodegradable plastic or through better material design to re-duce microplastic formation, such as developing textiles which shed fewer fibres. Using a life cycle thinking approach, one can take a more holistic view on the benefits which can be derived using various materials. Never the less, some plastic inevitably ends up as mismanaged waste, which directly enters the envi-ronment. Over time, this plastic can break down by sun irradia-tion and/or mechanical forces into ever-smaller pieces: mi-

croplastics. While microplastics have received a lot of attention in the mass media recently, during this presentation we will take a balanced view of the sources of (micro)plastic waste, how they are distributed in the environment, and what is known about their effects. Although scientific research on the topic has only begun intensifying recently, it is known that mi-croplastics can be found in many diverse environments, rang-ing from densely populated urban centers to remote beaches to the deep sea. While the occurrence of microplastic is seemingly high globally, in order to establish the level of risk for any envi-ronmental pollutant, one needs to not only consider exposure but also hazard. Determining adverse effects is not always easy or straight forward, so during the presentation we tackle the in which one can put the risks of plastic usage into context, and where environmental chemists and biologists currently stand on this issue.

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The continuous need for antibiotic stewardship in animal production and the response to a changing environment David C. Speksnijder DVM PhD 1,2 Jaap A. Wagenaar DVM PhD 1,3,4

1 Dept Infectious Diseases and Immunology, faculty of Veterinary Medicine, Utrecht University, the Netherlands 2 University Farm Animal Clinic ULP, Harmelen, the Netherlands3 Wageningen Bioveterinary Research, Lelystad, the Netherlands4 WHO Collaborating Center for Campylobacter and OIE Reference Laboratory for Campylobacteriosis

Antimicrobial resistance (AMR) is regarded one of the most serious threats for human and animal health in the near future if no action is undertaken. Although the contribution of antimi-crobial use (AMU) in animals to the AMR problem in human pathogens is difficult – if not impossible – to estimate, there is general consensus that AMU in humans, animals and plants, and the dissemination of resistant bacteria and antimicrobial residues must be controlled. This requires multidisciplinary ac-tions of all involved stakeholders at all levels.

At global level, the World Health Organization (WHO), Food and Agricultural Organization (FAO) and the World Organisa-tion for Animal Health (OIE) combine efforts in a ‘’One Health approach’’ to minimize the public health impact of AMR asso-ciated with AMU in farm animals. In response to a strong WHO urge in 2015, over 60 countries have by now developed and publicly shared a One Health National Action Plan describing each country’s specific approach to combat AMR. Putting pro-posed intervention measures in practice however faces several challenges as the drivers for AMR (i.e. overuse and misuse) differ greatly between production systems and between coun-tries.

Several examples exist of industrialized countries who have successfully reduced AMU and AMR rates over the last dec-ades. Scandinavian countries (Sweden, Denmark) have a long history of reducing and refining AMU in farm animals. In the Netherlands, which has a substantial livestock sector, over 65% reduction in AMU in livestock was achieved over the last 10 years. Amongst critical success factors were: clear AMU re-duction targets defined by the government, having full trans-parency and benchmarking on AMU/prescriptions per farm/veterinarian, the introduction of a 1-to-1 relationship between farmer and veterinarian and compulsory development of farm specific animal health plans and the existence of a surveillance system for AMR. Despite these considerable reductions in

AMU, there still seems room for improvement in specific areas. Further refinements in biosecurity, eradication of specific ani-mal diseases, boosting immunity of animals and finding alter-natives for specific antibiotic use practices are however still possible.

Artificial Insemination (AI) has been an enabling factor in the intensification of livestock production and is of great impor-tance to reduce the risk of animal disease transmission. To pre-serve ejaculates of farm animals (boars, steers) and stallions, cocktails of (often broad spectrum) antibiotics are being added as semen extenders to prevent spread of epizootics and to pre-vent growth of contaminant micro-organisms. Although effec-tive in lowering bacterial counts in semen, increasingly resist-ant bacteria are being found in ejaculates. The use of these antibiotics is mostly specified by government directives origi-nating several decades ago. In the light of prudent and restric-tive antibiotic use and increasing resistance levels to conven-tional antibiotics in semen extenders, it is necessary to critically re-evaluate this practice. A risk assessment including the risks of pathogens and their antimicrobial resistance pat-terns should be performed. As antimicrobials are also used as extender to reduce the concentration of non-pathogenic con-taminating bacteria, there is a challenge of predicting the re-sistance patterns of these bacteria. Another challenge is to perform the risk assessment European-wide with different lev-els of resistance. Sources to be used are the surveillance data in healthy animals and data on bacteria from diseased animals from diagnostic labs. When data are available, there has to be a translation for the specific use as semen extender (e.g. re-garding concentrations). One of the points to consider is the time and point of actions of antimicrobials: diluted samples are frozen immediately and used immediately after thawing. Final-ly, the use of antimicrobials should be in compliance with local regulations.

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Optimal Screening Tests of Semen Quality in early pubertal bulls destined for Artificial Insemination*C.M. O’ Meara a, C. Herrmann c, A. Kelly b, P. Lonergan b and B. Eivers a,a National Cattle Breeding Centre, Unit K4, M7 Business Park, Naas, Co Kildare, W91 WF59, Ireland.b School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Dublin, D04 N2E5, Ireland. c Institut Fur Fortpflanzung IFN Shonow, Bernauer Allee 10, 16321 Bernau bei Berlin, OT Schönow, Germany* Corresponding Author:

Dr Ciara O’ Meara National Cattle Breeding Centre Unit K4, M7 Business Park, Naas Co Kildare, W91 WF59, Ireland Tel: + 353 46 9541283 E-mail comeara@ncbc.ie

Accelerated genomic breeding programs result in more pres-sure on the Artificial Insemination (AI) Centres to collect, pro-cess and successfully freeze semen from young pubertal bulls. There currently are no single or combination of in vitro tests capable of consistently predicting field fertility and so most sires enter into test programs to determine their fertility based on non-return rates (NRRs) following AI in vivo. On average aprox 10% of bulls released into the field fall below acceptable levels for fertility. The aim of the current study was to deter-mine the most useful screening panel of in vitro test(s) capable of measuring potential fertility in pubertal bulls in order to pre-vent poor fertility bulls entering the program.

Young test sires (n=34) were genomically selected and semen was collected by Artificial Vagina. Semen was assessed for con-centration, volume, pre-freeze total motility and progressive motility and either rejected or frozen in 0.25 mL straws con-taining 15 x 106 sperm/mL. Semen was evaluated for total mo-tility and progressive motility at 0 and 120 min post-thaw to determine survival over time (Time Test). All ejaculates with total motility ≥50% at 0 and ≥40% at 120 min and progressive motility of ≥3 (scale 1 to 5) were released into the field and phenotypic pregnancy rates were estimated from both techni-cian and DIY inseminations. Rejection rates were assessed as the percentage of straws rejected over the total number of straws produced per bull. The top 3 and bottom 3 bulls from the panel of 34 bulls were selected based on phenotypic preg-nancy rate adjusted on a predictive model for external factors such as farm, technician effects (75+13%, 70+6% and 68+9% versus 51-5%, 46-10% and 33-15%) and semen was quantified for the biomarker proAKAP4 using ELISA assay. Morphology was assessed using Differential Interference Contrast (DIC) mi-croscopy. Thresholds for total normal forms and defects of the acrosome were classified as over 55% and <30%, respectively and assessed on mean values of frozen-thawed straws from random batches (n≥10 ejaculates/bull) from within the first

cohort of ejaculates released into the field for AI (n≈25 total ejaculates/bull). Comparisons for straws either rejected or ac-cepted based on semen parameters were firstly compared us-ing a mixed model of ANOVA in SAS. Then the bulls (n=26 fi-nal) were ranked on field fertility with a value of >60% assigned to high fertility and ≤60% assigned to low fertility bulls and groups were compared for all semen parameters measured.

At a cut-off value of 60% phenotypic pregnancy rate, the groups differed significantly with 71.0±0.50% and 47.4±7.05% phenotypic pregnancy rate for high and low groups, respec-tively (P<.0001). The mean age of bulls across all ejaculates was 456.1±7.2 days (range 375.9±8.8 to 630.1±5.0 days) and there was no difference in age between the high and low fertil-ity groups (P>0.05). However, there was an interaction be-tween age and straws rejected in the laboratory with a mean rejection rate per bull of 17.7±0.5% (range 0.0±0.0% to 65.6±0.9%). Ejaculates with lower concentrations of sperm/mL were more likely to be rejected at quality control checks (P=0.0036) and were significantly different between high and low groups (1190.4±22.87 versus 1100.7±32.29 x 109 sperm/mL, respectively; P=0.0239). Only midpiece defects appeared to increase with decreasing fertility (1.9±0.10% versus 2.8±0.15% for high versus low fertility groups respectively; P<0.0001) and were the most predictive parameter of field fer-tility (t value=-6.26). Protein levels for proAKAP4 varied with-in bulls (~43 ng/mL between individual ejaculates) and were significantly different between the 3 highest and 3 lowest fer-tility bulls (46.4±1.31 versus 69.1±1.15 respectively, P>0.05). Further studies are needed to understand why this protein is expressed in higher quantities in low fertility pubertal bulls compared to at high levels in high fertility mature bulls. This data suggests that both high incidence of midpiece defects and high levels of proAKAP4 are indicative of poor fertility out-come.

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Cattle Session II: Semen processing

M. bovis: PCR screening of the semen production.K. Elkjaer, K. Kupisiewicz and C. Alexandersen, VikingGenetics, Ebeltoftvej 16, 8960 Randers SØ, Denmark. Corresponding author: K. Elkjaer, kaelk@vikinggenetics.com

Mycoplasmas are the smallest and simplest prokaryotic cells that are capable of replicating (Nielsen, 2016). They have no rigid cell wall; instead, they are bound by a lipoprotein plasma membrane. The lack of cell wall makes mycoplasmas naturally resistant to some kinds of antibiotics.

The bacterium Mycoplasma bovis can be found as commensal on mucous membranes in cattle, e.g. in the udder, the respira-tory and genital tracts (Nielsen, 2016). In Denmark M. bovis has been isolated from clinical cases of mastitis, arthritis, pneu-monia and middle ear infections since the 1980’s . In Sweden and Finland, the bacterium has been isolated only recently in 2011-2012 (Evira, 2012; Lundberg, 2017).

In the period from 2011-2014 outbreaks were seen in Denmark, with 40-60 herds affected each year (Pedersen and Nielsen, 2018a). The reasons for these outbreaks remain uncertain, but genome sequencing of the M. bovis strains from the outbreaks shows, that these strains are closely related, and different from the previously isolated strains (Pedersen and Nielsen, 2018b). In 2016 a screening of bulk tank milk samples from all dairy herds in Denmark showed, that approximately 15% of herds tested positive for M. bovis. Only a few percent of these herds experienced outbreaks of mycoplasma.

Transmission of M. bovis is primarily via direct animal-to-ani-mal contact or via milking in the milking parlour (Areide et al., 2016). Recent research conducted in Finland showed the possi-bility of transmission of M. bovis via semen straws at AI (Haa-pala et al., 2018). In 2016, based on the preliminary results from this research, VikingGenetics took proactive actions and started screening of representative semen batches produced at VikingGenetics bull studs in Denmark. Test doses were pro-duced over a period of 25 weeks, giving in total more than 3000 semen batches obtained from different bulls. PCR meth-od of screening was originally developed by Thermo Fisher in Finland, but all the doses were tested by Eurofins. None of the tested batches were proven positive for M. bovis by PCR.

These findings are a small brick in the puzzle about M. bovis. Findings must be interpreted very cautiously as there still are several uncertainties, among others about the epidemiology and transmission between and within cattle herds. However, based on our screening, is seems that transmission of M. bovis with these semen doses produced at VikingGenetics bull sta-tions is quite unlikely.

References

Arede, M.; Nielsen, P.K.; Ahmed, S.S.U.; Halasa, T.; Nielsen, L.R.; Toft, N. (2015) A space-time analysis of Mycoplasma bovis: bulk milk antibody screening results from all Danish dairy herds in 2013-2014. Acta Vet. Scand. 58, 16.

Evira (2012). Mycoplasma bovis infection found in Finnish calves. Accessed 28.08.2018 from www.evira.fi: https://www.evira.fi/en/shared-topics/news/mycoplasma- bovis-infection-found-in-finnish-calves\

Haapala, V.; Pohjanvirta, T.; Vähänikkilä, N.; Halkilahti, J.; Simonen, H.; Pelkonen, S.; Soveri, T.; Simojoki, H.; Autio, T. (2018). Semen as a source of Mycoplasma bovis mastitis in dairy herds. Veterinary Microbiology, vol. 216 , pp. 60-66.

Lundberg, Å. (2017). Forskning för ökad kunskap om Mycoplasma bovis. Accessed 28.08.2018 from www.vxa.se: https://www.vxa.se/nyheter/2017/forskning-for-okad-kunskap- om-mycoplasma-bovis

Nielsen, L. R. (2016). Mycoplasma bovis pathogenesis, diagnostic methods and epidemiology of relevance for control and prevention. Paper presented at Finnish Annual Veterinary Congress 2016, Helsinki, Finland.

Pedersen, L. and Nielsen, L. R. (2018a). Historien om Mycoplasma bovis. Accessed 28.08.2018 from www.landbrugsinfo.dk: https://www.landbrugsinfo.dk/Kvaeg/Sundhed-og-dyrevelfaerd/Veterinaert-beredskab/mycoplasma/Sider/1-2-Historien-om-My-coplasma-bovis_note-2.aspx

Pedersen, L. and Nielsen, L. R. (2018b). Bakterien. Accessed 28.08.2018 from www.landbrugsinfo.dk: https://www.landbrugsinfo.dk/Kvaeg/Sundhed-og-dyrevelfaerd/Veterinaert-beredskab/mycoplasma/Sider/1-3-Bakterien_note-3.aspx

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Quality control: the multicolor assay and its biological relevanceEleni Malama, DVM, PhDClinic for Reproductive Medicine, Vetsuisse Faculty, University of Zurich, SwitzerlandE-mail: emalama@vetclinics.uzh.ch

The development of a simple methodology for the prospective approach of male fertility remains an issue of high interest in the field of animal biotechnology. The introduction of flow cy-tometry in animal andrology has enabled the fine examination of several aspects of sperm functionality. A wide variety of flu-orochromes with well-defined excitation and emission spectra, commonly referred to as “colors”, has been described for the assessment of sperm functional status. Plasma membrane and acrosome integrity, mitochondrial status, integrity of nuclear chromatin structure, oxidative stress and intracellular Ca2+ lev-els are typical examples of sperm attributes measurable through flow cytometry using one or two colors 1. Though widely used in other biomedical fields, flow cytometric assays employing three or more colors (henceforth referred to as mul-ticolor) are scarcely reported as part of sperm quality control schemes in sperm production centers.

The present study focuses on the following topics: a) the ad-vantages and limitations of multicolor flow cytometric assays against their one- or two-color analogues for the quality con-trol of commercially produced semen, b) examples of multi-color flow cytometric panels targeting key functions of mam-malian sperm, and c) the relevance of multicolor flow cytometry data to field fertility of commercially used sires.

Multicolor flow cytometry enables the analysis and quantifica-tion of sperm sub-populations that simultaneously exhibit three or more features; thus, it sheds light on the functional

heterogeneity of sperm as well as on the connections between sperm sub-populations with different functional attributes. In that direction, we developed a five-color flow cytometric pan-el, configured through a three-laser optical system, aiming at the simultaneous assessment of sperm plasma membrane and acrosome integrity, mitochondrial function and intracellular Ca2+ levels in commercially produced semen doses. A set of 91 frozen-thawed sperm samples collected from 19 commercially used sires was examined and the data of multicolor flow cyto-metric analysis were used for the retrospective prediction of field fertility of each sire (non-return rate 56 days after AI). The fertility status of approximately two thirds of the samples could be accurately predicted based on multicolor flow cytometric data. More interestingly, the deciding factor for predicting a sire’s fertility appeared to be the fraction of viable sperm (with intact plasma membrane and functional mitochondria) that could maintain low intracellular Ca2+ levels immediately after thawing, a piece of information that cannot be obtained through the commonly used one- or two-color flow cytometric assays.

In conclusion, the employment of a multicolor panel enables the combined assessment of the functional status of fro-zen-thawed sperm. Furthermore, there is strong indication that the functional heterogeneity of sperm, detectable through multicolor flow cytometry, is relevant to the fertilizing compe-tency of the sire in the field.

1 Petrunkina, A., Harrison, R., 2013. Fluorescence technologies for evaluating male gamete (dys)function. Reprod. Domest. Anim. 48, 11–24.

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Semen quality and field fertility of peripubertal bull ejaculatesM.L.W.J. BroekhuijseCRV B.V., Arnhem, The NetherlandsCorresponding author: marleen.broekhuijse@crv4all.com

The trend in the Artificial Insemination (AI) industry is to col-lect semen from bulls at a young age. With the use of genomic selection, and marketing semen globally, there is more pres-sure on even lowering this age to maximize the use of these bulls. Several parameters, like breed, body weight and nutri-tion affect the age at which a bull reaches puberty. After reach-ing puberty, an adjustment period follows before the ejaculates can be used for breeding (based on concentration, volume and quality of an ejaculate). It is well-known that young bulls have limited sperm quantity and quality characteristics. By lowering the age of the first collection, it is to be determined how this low age affects semen quality and fertility results.

Data from routine use in the AI program (CRV B.V., Arnhem, the Netherlands) were used, by collecting bull ejaculates at the barns in the Netherlands and processing the semen in the cen-tral laboratory into commercial insemination straws. Insemina-tion records (non return rate at 28 days (NR28%) and 56 days (NR56%) after insemination) were collected on individual se-men batch level and merged with the semen quantity and qual-ity records. Records from January 2013 till August 2018 were used (28.538 ejaculates). Of these records, for 14.463 ejacu-lates the fertility records were known. The mean age of the bulls at the moment of collection of an ejaculate was 30 ± 25 months, considering the whole database. When splitting the data, a decrease in age was seen over the years. In 2013, the mean age was still 36 months, while in 2017-2018 the mean age at collection was 26 months, being the first collections at 9-10 months of age.

When focusing on bull age < 24 months, the data from 2017 show an increase of +15% in post thaw semen motility (meas-ured by CASA), in the age period from 10 till15 months. Bulls > 15 months of age show on average a plateau value for post thaw semen quality. Similar pattern is shown for semen pro-duction (number of cells per ejaculate). This increase in semen quality is not shown in NR56%; the level only increases mini-mally during the first 2 years of age.

The effect of individual bull is known but must be considered when discussing age at ejaculation and the effect on semen quality and fertility. Although the overall mean shows an in-crease in semen quality in the first months of collection, there are bulls which do not show this impact when collected at young age: first ejaculates are immediately of good quality. Other bulls take more time to improve the level of post thaw semen quality, before the semen can be distributed.

Monitoring individual bulls by recording semen quality and fer-tility at individual semen batch level is a unique method to use young genomic bulls in the most efficient way. Known is the challenge to predict field fertility by general semen characteris-tics like post thaw semen motility; the correlations are low. Current AI bull management focuses on housing, nutrition, collection schedule and methods to optimize the use of young bulls. By proper AI bull management, by monitoring closely their field performance and by acting accordingly, the genomic potential of young bulls will be maximally used.

BETTER COWS | BETTER LIFE

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Pig Session II: New technologies: how do they fit in routine semen processing

Impact of transport stress on boar semen quality during long-term storageM. Schulze 1*; R. Bortfeldt 1, M. Jung 1, F. Fuchs-Kittowski 2* Corresponding author.

Tel.: +49 3338-709822 fax: +49 3338-709810 E-mail address: m.schulze@ifn-schoenow.de (M. Schulze)

1 Institute for the Reproduction of Farm Animals Schönow, Bernau, Germany2 University of Applied Science, Berlin, Germany

With the rapid development of artificial insemination (AI) of pigs over the last 20 years, the efficiency of boar semen usage has greatly increased. As a common practice, raw semen is di-luted with the aim to extend the longevity of the spermatozoa and increase the usability of boars of high genetic value to more than 50 semen doses per ejaculate. Increased require-ments for semen quality while simultaneously decreasing the number of spermatozoa per AI dose are driving sperm process-ing procedures in AI stations towards lab automation. Various exogenous components affect the boar sperm function during and after processing including, for example, the temperature and steps at which semen is diluted, the dilution procedure or the storage conditions. To date, not much is known about pos-sible shipping effects caused by vibration emissions and tem-perature. It can be assumed that, in addition to a stricter cen-tralization and professionalizing of boar semen processing in the future, optimizing boar semen shipping is of increasing importance.

The basic problem of identifying critical factors that could be improved during the shipping of boar semen can be resolved by real-time recording using a mobile sensing app in field trials. Modern smartphones contain several built-in sensors (such as accelerometer, digital compass, gyroscope, Global Positioning System (GPS), etc.) and built-in interfaces (Bluetooth, infrared, Wi-Fi) to connect external sensors (such as for temperature, etc.). These sensors can be used as measurement instruments enabling new applications across a wide variety of domains and give rise to a new area of research called mobile sensing. The aim of the present study was to collect shipping data using a special custom-programmed mobile sensing app (Transport-Log 1.0). This allowed for real-time recording of vibration emis-sions and temperature during boar shipping using various types of transportation vehicles on roads at different speeds

and on different surfaces. Furthermore, it was possible to iden-tify threshold values for vibration emissions that could interfere with semen quality and to develop new recommendations for boar semen shipping.

Field data were analyzed, transformed and used as standards for simulating vibration emissions by an orbital shaker (IKA MTS 4, Laborgeräte München) in a spermatology reference lab-oratory. Thirty ejaculates were collected randomly and diluted one-step isothermally in a split-sample procedure in a BTS ex-tender (Minitüb). The sperm concentration was adjusted to 23.5 x 106 spermatozoa ml-1. Semen was filled in 95 ml Quick-Tip Flexitubes® (Minitüb, Germany). The filling volume was 85 ± 1 ml. Samples were stored for seven days at 17°C. A compar-ison of the two main storage positions (horizontally vs. verti-cally) showed no influence on semen quality. In contrast, tem-perature undulations for 6 h below 10°C after processing affected semen quality, especially progressively motile, acro-some-defect, mitochondrially active and membrane-intact spermatozoa. Temperature undulations between 10°C and 30°C showed no effect. Finally, we found that maximal vibra-tion emissions with circular horizontal frequencies of 300 rpm for 6 h during shipping had a negative impact on pH value of the BTS extender and semen quality during long-term storage. This study provides valuable knowledge for shipping of boar semen in the AI industry. A new monitoring tool for boar semen shipping was established using mobile sensing. Mobile sensing makes it easier for users to accept and use an application as it utilizes technology and equipment that many people already own. Finally, our long-term goal is to find the optimal transport packaging for boar semen AI doses, which provides the neces-sary protection from mechanical stress during short-term and long-term transport. Supported by FBF Germany.

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In the last years a large improvement in informatic technolo-gies (IT) could be seen. The computers and server became very fast and the development of web services allows to change data between different databases or other software systems. This offers the opportunity to use IT even in semen processing units for fresh boar semen, were the in-time production is com-mon. The use of IT in a routine lab can help to improve semen quality and work flow.

GFS practices some new developments concerning identity in-surance, data transfer via web services and monitoring of Pro-duction parameters.

Chip identChip ident improves the safety concerning the correct identity of the ejaculate. Each boar is chipped with a transponder. Im-mediately after semen collection the transponder is read with a “Psion” reading system. In the next step the Psion initiates a printer. With this printer a bar code label is printed with infor-mation to the identity of the boar, his body temperature and libido score. And last but not least the identity of the techni-cian, who did the collection. This label then is fixed on the se-men collection jar before the semen is sent to the lab.

Barcode reading at each quality control positionIn the semen processing room (lab) where quality control and semen processing occur, at each position of control or process-ing a bar code reader and a computer is established. Via read-ing the barcode, the correct identity of the ejaculate is ensured. In the first step at each position the barcode is read. The system finds the right boar and shows it in the user interface.

Scale (weight/volume) and colorimeter (density) as well as CASA system, dilution-system and filling machine is connected with its local computer or gets automatically information from the main Lab software.

The measured values are transferred to the local computer. From this the data are sent via a web service to the server of the main lab software. After doing the quality control the next step is reading the bar code into the main lab software. In the main lab software interface, the user can see all data, collected up to now on the screen. The user than has to give input about colour and smelling of the ejaculate. The software automatical-ly decides, if the ejaculate can be used or not and calculates the number of doses to produce on the base of all quality measure-ments.

When you read the barcode at the dilution system, the number of portions is available there and the correct amount of diluter is filled into the jar. The number of portions to be produced is also sent to the filling machine.

Data transfer via web servicesTwo big advantage of using webservices for data transfer have to be pointed out: First: with this system you do not have to process the ejaculates in the correct sequence. You are allowed to switch the order around. Second: The information can be sent over large distances. The server of the main lab software can be in the headquarters of the AI organisation. The informa-tion of all semen data even from different labs are available in the headquarters just in time.

Documentation of the time of measurement of each ejac-ulate in each position of quality control and processingSome processes in the lab are time sensitive, for example put-ting extender (pre-extender) to the raw ejaculate to stop bac-terial growth and stabilise the membranes of the semen cells. To have a control about these time steps, the moment of meas-urement of each ejaculate in each production step is fixed in the database and can be used for controlling and improving the workflow.

Statistic programs help to analyse and visualise produc-tion dataA statistic program which visualises data in figures helps to control semen data and the work flow. The instrument allows to evaluate all significant values with the aim of optimizing se-men output, semen quality and work flow.

Modern technics to improve semen quality and laboratory managementDr. Sabine Brüning, GFS

28

Reducing antibiotics in boar semen extendersChristin Oehler 1, Prof. Dr. Fredi Janett 1, Dr. Sarah Schmitt 2, Dr. Eleni Malama 1, Prof. Dr. Heinrich Bollwein 11 Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland2 Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

With emerging resistances in bacterial populations against commonly used antibiotics the prudent use of such is consist-ently subject to discussion, not only in human medicine but also in particular in livestock associated fields like the pig in-dustry. Antimicrobial resistances in the final dose of liquid stored boar semen is a known problem and oftentimes caused by a lack of hygiene during the production process in the lab (Schulze et al. 2015). Up until now it is compulsory for boar semen extenders to contain antibiotics in order to prevent dis-eases (OIE, 2016; EU Directive 90/429/EEC), but in order to prevent further tightening of the situation already years ago first studies were carried out in order to replace or reduce these substances. These studies were based on different approaches like lowering storage temperatures or searching for alternative substances. Our attempt was based on the development of a new flow cytometric technique to enable a fast and cost-effi-cient enumeration of the total viable bacteria count in native boar ejaculates as a first step. This was then followed by stor-age trials to determine a cut-off value for the critical amount of bacteria which leads to a decrease of sperm quality when anti-biotic-free extender is used.

The flow cytometric method is based on a staining consisting SYBR Green I and Propidium Iodide in order to determine the total viable count (TVC) of bacteria. It was validated in two steps by analyzing a total of 224 fresh boar ejaculates, first by spiking the ejaculates with defined numbers of bacteria com-monly detected in boar semen, and second by performing measurements in parallel with a classical bacteriological cul-ture technique and the new flow cytometric method. In the first part a strong correlation between the detected and expected numbers could be observed (r = 0.96; P < 0.001), while in the second part of the study the TVC determined by both methods correlated only moderately (r = 0.28; P < 0.01; median MPN: 24,000 ± MAD 21,600 bacteria/mL; median flow cytometry: 24,426 ± MAD 15,610 bacteria/mL).

For the third part of the study a total of 103 ejaculates were collected from Duroc and Premo® boars of two AI-stations. Equal parts of semen were extended (Androstar® Plus, Mini-tube, Germany) with and without addition of AB (Eurococktail, Minitube, Germany). Sperm characteristics and bacterial con-tent were determined daily using CASA and flow cytometry during 6 days of storage (18 °C). There were no differences (P≥0.05) in sperm characteristics in the first 2 days after collec-tion and dilution of sperm with and without AB. At 2 and 4 days of storage moderate negative correlations (-0.25<r<-0.50, P<0.05) between bacterial count and the percentage of plasma membrane- and acrosome-intact sperm could be found in the group without AB, while in the group with AB there were no relationships (P≥0.05) between these parameters. From day 4 on bacterial count was higher (P<0.05) in the group without AB. The absence of AB had no negative effects (P≥0.05) on motility and mitochondrial membrane potential of sperm dur-ing the whole experiment.

In summary the developed flow cytometric method enables a fast determination of the total viable bacteria count in native boar ejaculates. Increasing resistances against antibiotics is a challenging task not only in livestock associated areas such as the pig industry. The presented approach is one possible way to cut down on the use of antibiotics in boar semen extender, nev-ertheless further studies have to follow to ensure that there is no negative effect on semen quality nor on animal welfare by potential spread of diseases.

References:

Schulze, M.; Ammon, C.; Rudiger, K.; Jung, M.; Grobbel, M. (2015): Analysis of hygienic critical control points in boar semen production. In: Theriogenology 83 (3), S. 430–437.

MinitubeArtificial Reproduction Technologies

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MinitubeArtificial Reproduction Technologies

www.minitube.com

Peace of mind

Time saving

Efficient filling and sealing

Smart ergonomics

Unique traceability

• Fertility comparable with conventional semen

• Doubles the eff ect of genomic selection

• Females from your best cows

• Increased production

• Easier calvings

SexedULTRA

Peace of mind

Time saving

Efficient filling and sealing

Smart ergonomics

Unique traceability

• Fertility comparable with conventional semen

• Doubles the eff ect of genomic selection

• Females from your best cows

• Increased production

• Easier calvings

SexedULTRA

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