A dual colour FISH method for routine validation of sexed Bos taurus semen

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.Peer reviewe

Veise sigimine: kõrgkooliõpik

Eestis on veisekasvatusest lugu peetud juba kiviajast peale. Meil on heade tõuomadustega ja suure toodanguga piimakari ja kiiresti kasvav lihakari ning me suudame toota kvaliteetset piima ja veiseliha nii kohalikele tarbijatele kui ka ekspordiks. Veiste sigimisprobleemid on oluliseks veisekasvatuse kasumlikkust mõjutavaks teguriks. Selleks, et sigimisprobleemide olemust mõista ja nende teket ennetada või neid ravida, on vajalikud põhjalikud teadmised veise sigimise erinevatest aspektidest. Seni puudub üks terviklik veise sigimise alast teavet koondav eestikeelne õppevahend. Käesoleva õpiku abil soovime seda lünka täita. Oleme õpikusse kokku kogunud teadmised suguorganite morfoloogiast ja füsioloogiast, tiinusest ja sünnitusest ning poegimisjärgsetest haigustest. Suguorganite anatoomiat käsitletakse õpikus mitte klassikalise, vaid funktsionaalse anatoomia seisukohast. Veise tiinuse ja sünnituse

A dual colour FISH method for routine validation of sexed Bos taurus semen

Abstract Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples

Veise sigimine : kõrgkooliõpik

KõrgkooliõpikÜlle Jaakma ja Mihkel Jalakase toimetatud õpik „Veise sigimine“ valiti 2018. aasta parimaks eestikeelseks kõrgkooliõpikuks.Eestis on veisekasvatusest lugu peetud juba kiviajast peale. Meil on heade tõuomadustega ja suure toodanguga piimakari ja kiiresti kasvav lihakari ning me suudame toota kvaliteetset piima ja veiseliha nii kohalikele tarbijatele kui ka ekspordiks. Veiste sigimisprobleemid on oluliseks veisekasvatuse kasumlikkust mõjutavaks teguriks. Selleks, et sigimisprobleemide olemust mõista ja nende teket ennetada või neid ravida, on vajalikud põhjalikud teadmised veise sigimise erinevatest aspektidest. Seni puudub üks terviklik veise sigimise alast teavet koondav eestikeelne õppevahend. Käesoleva õpiku abil soovime seda lünka täita. Oleme õpikusse kokku kogunud teadmised suguorganite morfoloogiast ja füsioloogiast, tiinusest ja sünnitusest ning poegimisjärgsetest haigustest. Suguorganite anatoomiat käsitletakse õpikus mitte klassikalise, vaid funktsionaalse anatoomia seisukohast. Veise tiinuse ja sünnituse patoloogia osas antakse ülevaade ka väärarendite ja abordi problemaatikast. Esmakordselt käsitletakse eestikeelses õpikus kaasaegset sigimise biotehnoloogiat, sh embrüotehnoloogiat ja transgeenset tehnoloogiat, koos võimalike rakendustega aretustöös. Õpik on mõeldud eeskätt veterinaarmeditsiini eriala üliõpilastele, aga seda saavad käsiraamatuna kasutada ka praktiseerivad loomaarstid, seemendustehnikud ja loomakasvatuse spetsialistid. Lisaks sellele saavad siit informatsiooni bioloogia eriala üliõpilased ja biotehnoloogia ning kliinilise diagnostika spetsialistid, samuti bioloogiaõpetajad. Õpiku autorid on õppejõud ja teadlased, kes on oma kitsama valdkonna tunnustatud asjatundjad ning puutuvad oma töös käsitletud teemade ringi puudutavate probleemidega kokku iga päev. Täname kõiki autoreid nende suure panuse eest õpiku valmimisse. Suur tänu Eha Järvele, kes on rahulikult ja asjatundlikult aidanud eri autorite kirjutatud osad üheks tervikuks ühendada. Oleme tänulikud kõigile kolleegidele, kes oma tähelepanekute ja soovitustega on selle raamatu valmimisel kaasa aidanud. Loodame, et õpik pakub nii õppuritele kui ka praktikutele kasulikku ja vajalikku informatsiooni. Ülle Jaakma õpiku toimetaj

Assessment of sperm attributes of frozen-thawed AI doses from Swedish and Estonian dairy bull sires

The fertility of bull semen used in artificial insemination (AI) is essential for the effective use of this technology in bovine breeding. The laboratory evaluation of semen from healthy bulls, before and after freezing for AI, is largely based on subjectively scoring sperm motility, and on measurement of sperm concentration. Since the relationship between these parameters is barely indicative of the fertility of the semen samples, other, often more complicated methods are used to evaluate a battery of sperm attributes of importance for their fertilizing capacity. This thesis evaluated the usefulness of various conventional and novel sperm quality tests in assessing frozen-thawed (FT) AI doses produced from semen of young, unproven and older, progeny-tested dairy bulls of the Estonian Holstein Friesian (EHF) and Swedish Red and White (SRB) breeds and their relationship with field fertility after AI. In addition, the influence of sperm preparation methods prior to quality measurements was investigated. Semen AI doses were evaluated immediately post-thaw (PT) and after cleansing through washing/resuspension (W) and swim-up (SU). Special attention was given to age-related changes in semen from 1- v. 4-, and 3- v. 5- and 7-year-old bulls. Spermatozoa were evaluated for their morphology (microscopy on wet and dried, stained smears), motility (assessed subjectively and by computer-assisted sperm analysis [CASA]) and membrane integrity (using SYBR-14/propidium iodide [PI] fluorophores and microscopy). Mitochondrial activity (with MitoTracker Deep Red), membrane fluidity (using Merocyanine 540 lipid dye/Yo-Pro-1/H332) and deoxyribonucleic acid (DNA) integrity (using acridine orange [AO] staining) were assayed using flow cytometry (FC). Use of SU provided spermatozoa with significantly better motility, morphology, membrane integrity, mitochondrial activity and chromatin stability compared with either PT or W treatment. Although the SU selection step increased the proportion of spermatozoa with a stable plasma membrane, it also initiated membrane destabilization. Age differences in sperm quality (motility, membrane integrity and mitochondrial function) were seen PT (at 4 years of age for SRB and at 3–7 years for EHF bulls) and were accentuated when using SU, but not W, as pre-selection procedure. No changes in chromatin stability were, however, registered for either breed. Only few FT sperm quality attributes (e.g. average path velocity (VAP), proportion of cells with tail abnormalities, and non-linear motility) showed significant correlation to fertility after AI when evaluated PT or after W. More attributes (e.g. CASA total sperm motility, concentration of motile and linearly motile spermatozoa, VAP, as well as the percentage of spermatozoa with unstable plasmalemma) had a significant relationship with non-return rate (NRR) when spermatozoa were examined after SU. In conclusion, SU was superior to W in harvesting intact spermatozoa with attributes essential for fertilization, distinguishing bulls and revealing age-dependent changes. Also, the use of CASA and FC of fluorophore-loaded spermatozoa increased the objectivity of the tests assayed. Combining SU and CASA, methods considered to be easily applicable at the semen-producing enterprises, indicated that the overall semen quality of proven bulls was predictable from measurements done at an early age. While sperm membrane stability and mitochondrial activity were seen as suitable markers for monitoring semen quality, measurements of chromatin intactness could not yield additional information

Identification of bull semen microbiome by 16S sequencing and possible relationships with fertility

Reports on the use of 16S sequencing for the identification of bacteria in healthy animals are lacking. Bacterial contamination of bull semen can have a negative effect on the sperm quality. The aims of this study were threefold: to identify bacteria in the semen of healthy bulls using 16S sequencing; to investigate the differences in the bacterial community between individual bulls; and to establish if there was a relationship between the bacteria isolated and bull fertility. Semen from 18 bulls of known fertility was used for the DNA extraction and 16S sequencing; 107 bacterial genera were identified. The differences in the amplicon sequence variants (ASVs) and the numbers of genera between bulls were noted. Negative correlations (p < 0.05) between several bacterial genera with Curvibacter, Rikenellaceae RC9-gut-group and Dyella spp. were seen. Other negatively correlated bacteria were Cutibacterium, Ruminococcaceae UCG-005, Ruminococcaceae UCG-010 and Staphylococcus, all within the top 20 genera. Two genera, W5053 and Lawsonella, were enriched in bulls of low fertility; this is the first time that these bacteria have been reported in bull semen samples. The majority of the bacteria were environmental organisms or were species originating from the mucous membranes of animals and humans. The results of this study indicate that differences in the seminal microbiota of healthy bulls occur and might be correlated with fertility

Identification of Bull Semen Microbiome by 16S Sequencing and Possible Relationships with Fertility

Reports on the use of 16S sequencing for the identification of bacteria in healthy animals are lacking. Bacterial contamination of bull semen can have a negative effect on the sperm quality. The aims of this study were threefold: to identify bacteria in the semen of healthy bulls using 16S sequencing; to investigate the differences in the bacterial community between individual bulls; and to establish if there was a relationship between the bacteria isolated and bull fertility. Semen from 18 bulls of known fertility was used for the DNA extraction and 16S sequencing; 107 bacterial genera were identified. The differences in the amplicon sequence variants (ASVs) and the numbers of genera between bulls were noted. Negative correlations (p &lt; 0.05) between several bacterial genera with Curvibacter, Rikenellaceae RC9-gut-group and Dyella spp. were seen. Other negatively correlated bacteria were Cutibacterium, Ruminococcaceae UCG-005, Ruminococcaceae UCG-010 and Staphylococcus, all within the top 20 genera. Two genera, W5053 and Lawsonella, were enriched in bulls of low fertility; this is the first time that these bacteria have been reported in bull semen samples. The majority of the bacteria were environmental organisms or were species originating from the mucous membranes of animals and humans. The results of this study indicate that differences in the seminal microbiota of healthy bulls occur and might be correlated with fertility