Normal Bulls Research Articles

A potent allele marker related to low bull conception rate in Japanese Black bulls

Over the years, there has been considerable variation in the bull conception rate (BCR) of Japanese Black cattle; moreover, several Japanese Black bulls with a low BCR of ≤10% have been identified. However, the alleles responsible for the low BCR are not determined yet. Therefore, in this study, we aimed to identify single-nucleotide polymorphisms (SNPs) for predicting low BCR. To this end, the genome of Japanese Black bulls was comprehensively examined by a genome-wide association study with whole-exome sequencing (WES), and the effect of the identified marker regions on BCR was determined. The WES analysis of six sub-fertile bulls with a BCR of ≤10% and 73 normal bulls with a BCR of ≥40% identified a homozygous genotype for low BCR in Bos taurus autosome 5 in the region between 116.2 and 117.9 Mb. The g.116408653G > A SNP in this region had the most significant effect on the BCR (P-value = 1.0 × 10−23), and the GG (55.4 ± 11.2%) and AG (54.4 ± 9.4%) genotypes in the SNP had a higher phenotype than the AA (9.5 ± 6.1%) genotype for the BCR. The mixed model analysis revealed that g.116408653G > A was related to approximately 43% of the total genetic variance. In conclusion, the AA genotype of g.116408653G > A is a useful index for identifying sub-fertile Japanese Black bulls. Some positive and negative effects of SNP on the BCR were presumed to identify the causative mutations, which can help evaluate bull fertility.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

BackgroundThe homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.ResultsBy introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.ConclusionThe allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template.This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Not gone but forgotten: Tritrichomonas foetus in extensively-managed bulls from Australia’s Northern Territory

Bovine trichomonosis, caused by infection with the protozoan parasite Tritrichomonas foetus, is globally recognised as a cause of reproductive failure in cattle. Maintained in clinically normal bulls, T. foetus infection results in infertility and abortion in infected cows. In Australia’s Northern Territory (NT), logistical limitations associated with extensive livestock production inhibit wide-scale testing and diagnosis, allowing the parasite to persist undetected. In the present study, T. foetus was detected in 18/109 preputial cultures collected from bulls on a property in the NT with a history of low birth rates and reproductive failure using real-time PCR testing. Of the T. foetus-positive samples, 13/18 were genotyped using the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S rDNA unit. Selected samples were further characterised using the protein-coding genes of cysteine proteases (CP-1, 2, 4–9) and cytosolic malate dehydrogenase 1 (MDH-1) to determine if the isolates were ‘bovineʼ, ‘felineʼ or ‘Southern Africaʼ genotypes. All samples were 100% identical to the T. foetus ‘bovine’ genotype across all markers. This is the first reported case of trichomonosis in Australian cattle since 1988 and is a reminder that T. foetus should be considered whenever reproductive failure occurs in extensive cattle systems.

Effect of cattle-specific estrus molecules on libido and semen production of zebu bulls under tropical climate.

Zebu bulls are a shy breeder and they exhibit optimum libido in the presence of females with estrus phase. Continuous semen collection with the use of male dummy leads to lack of adequate sexual stimulation. Therefore, the present study was designed to test the effect of estrus-specific molecule(s) for effective sexual preparation of donor bulls. The bulls were divided into normal and poor libido group, five bulls in each group by taking 1-month control study data after collecting the information of individual bull's sexual behaviour during semen collection by regular semen collector. The bulls were never being exposed to female animals and semen was collected by an artificial vagina. The ten animals were exposed to a glycerol-water solution (50/50 v:v) as control and then exposed to estrus-specific molecules one by one. The estrus-specific molecules like squalene, 1-iodoundecane, acetic acid, coumarin, propionic acid, oleic acid, and 2-butanone were purchased from Sigma-Aldrich Company, USA, and the molecules were solubilised individually in a non-pressurised aerosol dispenser as 1.0% concentration in glycerol-water solution (50/50, v:v). Identical bulls were used as the control and exposed to each molecule one by one by giving a refractory period of 14days. A nasal spray of acetic acid or 2-butanone significantly (p < 0.05) reduced reaction time (RT) and total time taken to ejaculate (TTTE) in normal libido bull group. Semen volume, sperm concentration, and the total number of sperm per ejaculation obtained did not show significant improvement in the normal libido group of bulls after the application of estrus-specific molecules as compared to the control. In poor libido group, acetic acid, oleic acid, and 2-butanone application showed significant (p < 0.01) improvement in RT and TTTE as compared to the control group, whereas semen production variables like sperm concentration and total sperm output per ejaculation increased significantly (p < 0.05) except semen volume. There was significant (p < 0.01) reduction in RT (%) and TTTE (%) after the application of acetic acid followed by 2-butanone and oleic acid. The sperm concentration and total sperm output per ejaculation were more after the application of each molecule but significant increase (p < 0.05) in sperm concentration was observed with 2-butanone (11.42%), acetic acid (11.42%), and oleic acid (10.13%), whereas total sperm output per ejaculation increased significantly (p < 0.05) only after the application of acetic acid and 2-butanone (24.75% and 26.84%). Hence, it can be concluded that acetic acid, 2-butanone, and oleic acid are effective for better sexual preparation of Sahiwal bulls and total sperm output per ejaculation.

Serum and seminal plasma IGF-1 associations with semen variables and effect of IGF-1 supplementation on semen freezing capacity in buffalo bulls

The objective of the study was to establish correlation of seminal and serum IGF-1 with seminal attributes, estimate antioxidant potential of IGF-1 by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays and to study the effect of IGF-1 supplementation on semen cryopreservation. For this study, buffalo bulls were divided into sub-fertile (n = 2) and normal (n = 5) on the basis of sperm mass motility and individual motility. The serum IGF-1 concentration of normal bulls was greater than in sub-fertile bulls, but there was no difference in the seminal IGF-1 concentration among the groups. The values from correlation analyses indicated that serum IGF-1 concentration is positively correlated with semen mass motility and sperm concentration. In the second experiment, IGF-1 did not have antioxidant activities when assessed with DPPH and FRAP assays. In the third experiment, the ejaculates of normal and sub-fertile bulls were cryopreserved using semen extender in which there was IGF-1 supplementation at 0 (control), 50, 100, 150, 200, 250, 350 and 450 ng/mL of extender. Supplementation of IGF-1 at 250 ng/ml resulted in improved sperm motility, longevity and membrane intactness as compared to control after cryopreservation of semen from normal buffalo bulls, but not sub-fertile bulls. In summary, serum IGF-1 concentration was correlated with sperm mass motility and concentration in buffalo bulls and supplementation of IGF-1 protected sperm during the cryopreservation process but effects were not due to direct antioxidant activity.

Improvement of sexual behavior and semen quality by therapeutic approach and zinc supplementation on Karan Fries

Sub-fertility is one of the major concerns in crossbred bulls as 42.98% bulls are getting culled due to sub- fertility problems therefore, a study was planned on 18 Karan Fries bulls producing poor quality semen or not mounted. They were randomly grouped into 3 treatment groups of 6 animals each and 2 normal bulls were kept as control. Sexual behavior and semen quality were studied during pre-therapy (1 month), therapy (90 days) and post- therapy (90 days) period. Data were analyzed using ANOVA. During therapy period, first group treated with 10 g of herbal product orally, second group with 0.0082 mg of GnRH intramuscularly at 10 days interval and third group with 80 ppm of zinc for 90 days for each group. The results depicted that herbal and GnRH treatment significantly reduced the reaction time, whereas herbal, GnRH treatment and zinc supplementation significantly improved the libido score and reduced per cent of non-mounts during the therapy and post-therapy period. The volume of semen and level of testosterone improved in all treatments groups. Herbal, GnRH treatment and zinc supplementation increased the average number of good and medium quality ejaculates/month/bull and maximum efficiency achieved during the treatment period. There was increase in percent live sperm and decrease in abnormality in herbal treated and zinc supplemented group. Therefore, it can be concluded that GnRH therapy, herbal treatment and zinc supplementation will be effective to overcome the sub-fertility problem in crossbred bulls.

22 SEMEN AND REPRODUCTIVE PROFILES OF CLONED ANATOLIAN GREY CATTLE

Anatolian grey cattle (endangered native Anatolian cattle) as 1 male (clone 1) and 4 females (clones 2–5) were produced from cells of 1 male and 1 female cattle by somatic cell nuclear transfer (SCNT) in a previous study. In this study, we examined the reproductive potential of these cloned animals, which are now 4 and 5 years old. The parameters evaluated by phase contrast microscopy for motility, TUNEL for DNA fragmentation, eosin staining for viability, Hoechst 33258 staining and hypo-osmotic swelling test (HOST) for membrane integrity, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) for acrosome integrity of frozen-thawed spermatozoa, as well as birth and survival of calves following insemination with frozen-thawed semen of cloned and nuclear donor bull and normal bull. Six ejaculates and 3 samples per ejaculate from each bull were tested, and the Mann-Whitney U test was used to analyse the data. The spermatological parameters of cloned bull semen – volume, concentration, and motility of fresh – were within accepted limits for artificial insemination (4.60 ± 0.47 mL, 1.55 ± 0.21 × 109 spermatozoa mL–1, 80.00 ± 1.07%, respectively). Frozen-thawed sperm motility and viability rate were higher in the cloned bull (56.6%, 56.7%) than in its nuclear donor (47%, 43%; P &lt; 0.05). Intact membrane and DNA fragmentation rate of cloned bull and its nuclear donor bull sperm were similar (P &gt; 0.05) but the intact acrosome rate of cloned bull was higher than that of its nuclear donor (P &lt; 0.05). Low rates in frozen-thawed sperm of nuclear donor can be related to storage time of sperm which were frozen 5 years before. One (clone 4) of the cloned grey heifers was artificially inseminated with frozen semen from nuclear donor bull and the other (clone 5) was naturally mated with a Holstein bull. Two healthy calves were delivered naturally. When same cloned cows (clones 4–5) and 2 other cloned heifers (clones 2–3) were artificially inseminated with frozen semen of the cloned grey bull, clones 2 and 4 gave birth to 2 healthy female calves. One cloned cow (clone 3) aborted in the third month of gestation and other one (clone 5) is currently 8 months pregnant. Two calves of clone 4 and 5 are 17 months old and 2 other calves of clone 2 and 4 are now 6 and 1 months old. Except for clone 3, our results show that cloned Anatolian grey bull and cows produced from frozen cells in gene bank have normal fertility.

Fine mapping for Weaver syndrome in Brown Swiss cattle and the identification of 41 concordant mutations across NRCAM, PNPLA8 and CTTNBP2.

Bovine Progressive Degenerative Myeloencephalopathy (Weaver Syndrome) is a recessive neurological disease that has been observed in the Brown Swiss cattle breed since the 1970’s in North America and Europe. Bilateral hind leg weakness and ataxia appear in afflicted animals at 6 to 18 months of age, and slowly progresses to total loss of hind limb control by 3 to 4 years of age. While Weaver has previously been mapped to Bos taurus autosome (BTA) 4∶46–56 Mb and a diagnostic test based on the 6 microsatellite (MS) markers is commercially available, neither the causative gene nor mutation has been identified; therefore misdiagnosis can occur due to recombination between the diagnostic MS markers and the causative mutation. Analysis of 34,980 BTA 4 SNPs genotypes derived from the Illumina BovineHD assay for 20 Brown Swiss Weaver carriers and 49 homozygous normal bulls refined the Weaver locus to 48–53 Mb. Genotyping of 153 SNPs, identified from whole genome sequencing of 10 normal and 10 carrier animals, across a validation set of 841 animals resulted in the identification of 41 diagnostic SNPs that were concordant with the disease. Except for one intergenic SNP all are associated with genes expressed in nervous tissues: 37 distal to NRCAM, one non-synonymous (serine to asparagine) in PNPLA8, one synonymous and one non-synonymous (lysine to glutamic acid) in CTTNBP2. Haplotype and imputation analyses of 7,458 Brown Swiss animals with Illumina BovineSNP50 data and the 41 diagnostic SNPs resulted in the identification of only one haplotype concordant with the Weaver phenotype. Use of this haplotype and the diagnostic SNPs more accurately identifies Weaver carriers in both Brown Swiss purebred and influenced herds.

Two dimensional SDS PAGE analysis of epididymal tissue proteins of normal and castrated bulls (Bos taurus) and identification of an androgen dependant protein

A study was undertaken with an objective of two dimensional SDS-PAGE analysis of protein profile in tissues of all caput, corpus and cauda of epididymis in both castrated and normal bulls and identification of androgen dependent protein/s in the epididymal tissues of bull (Bos taurus). Two dimensional SDS-PAGE analysis for protein spots of different molecular weight (MW) with pI 3.5 to 7.35 and pI > 7.35 to 9.3 between the three regions in the normal and castrated bull as well as between normal and castrated bull of a particular region revealed significant differences. Similarly, comparison between the three regions of the normal and castrated bull epididymis for the number of protein spots irrespective of the MW in pI range of 3.5 to 7.35 and pI range > 7.35 to 9.3 revealed significant differences. The number of protein spots found to be significantly higher in caput, corpus and cauda epididymis of the normal bull when compared to similar region of the castrated bull epididymis. Most of the proteins, which are secreted, are having MW between 20 and 85 kDa. Six proteins, which are known to be highly dependent on androgens, are also of acidic in nature except for one protein having basic pH. A protein spot (pI: 6.55–6.85, and MW: <20 kDa) which appeared at the same site in all the three regions of the epididymis in the normal bull but was absent at the same site in all the three regions of the castrated bull was subjected for identification of the protein by MALDI-MS. The results revealed that this protein is an interferon-stimulated protein and it could be ISG15/UCRP.

Identification of an FBN1 mutation in bovine Marfan syndrome‐like disease

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause Marfan syndrome (MFS). Frequent observation of cattle with a normal withers height, but lower body weight than age-matched normal cattle, was recently reported among cattle sired by phenotypically normal Bull A, in Japanese Black cattle. These cattle also showed other characteristic features similar to the clinical phenotype of human MFS, such as a long phalanx proximalis, oval face and crystalline lens cloudiness. We first screened a paternal half-sib family comprising 36 affected and 10 normal offspring of Bull A using the BovineSNP50 BeadChip (illumina). Twenty-two microsatellite markers mapped to a significant region on BTA10 were subsequently genotyped on the family. The bovine Marfan syndrome-like disease (MFSL) was mapped onto BTA10. As FBN1 is located in the significant region, FBN1 was sequenced in Bull A, and three affected and one normal cattle. A G>A mutation at the intron64 splicing accepter site (c.8227-1G>A) was detected in 31 of 36 affected animals (84.7%). The c.8227-1G>A polymorphism was not found in 20 normal offspring of Bull A or in 93 normal cattle unrelated to Bull A. The mutation caused a 1-base shift of the intron64 splicing accepter site to the 3' direction, and a 1-base deletion in processed mRNA. This 1-base deletion creates a premature termination codon, and a 125-amino acid shorter Fibrillin-1 protein is produced from the mutant mRNA. We therefore conclude that the c.8227-1G>A mutation is causative for MFSL. Furthermore, it was suggested that Bull A exhibited germline mosaicism for the mutation, and that the frequency of the mutant sperm was 14.9%.

Livability of albino and normal bull spermatozoa at 5 degrees C in citrate, milk and tris yolk extenders.

Summary Twenty ejaculates each, of an albino bull of the Brown Swiss breed and a normal Holstein Friesian bull were diluted with citrate-yolk, milk-yolk, and Tris (hydroxymethyl) aminomethane-yolk extenders and preserved at 5 °C. Considering the average sperm motility in the diluted samples, the Tris-yolk extender was superior to the milk-yolk and citrate-yolk extenders and the milk-yolk extender was superior to the citrate-yolk extender. No significant difference was found between the percentage of motile spermatozoa in the diluted samples of the albino and normal bull. Zusammenfassung überlebenszeit von normalen Bullenspermien bei 5 °C in Citrat- Milch- und Tris-Eigelbverdünnern Von einem Albinobullen (Brown Swiss) und einem normalen Bullen (Holstein-Friese) wurden je 20 Ejakulate mit Citrat-Eigelb, Milch-Eigelb und Tris (Hydroxymethyl) Aminomethan-Eigelb verdünnt und bei 5 °C aufbewahrt. Auf Grund der Spermienmotilität erwies sich der Tris-Eigelbverdünner besser als der Milch-Eigelb und der Citrat-Eigelbverdünner. Der Milch-Eigelbverdünner war besser als der Citrat-Eigelbverdünner. Die Beweglichkeit der Spermien des Albino- und Normalbullen zeigte keinen signifikanten Unterschied. Résumé Temps de survie du sperme normal de bovin à 5 °C dans un diluant au jaune d'oeuf avec du citrate, du lait et du «Tris» 20 éjaculats pour chacun d'un taureau albinos (Brown Swiss) et d'un taureau normal (Holstein-Friese) ont été dilués dans du jaune d'oeuf avec du citrate, du lait, du «Tris» (hydroxymethyl) aminométhane et conservés à 5 °C. Le jaune d'oeuf plus «Tris» s'est révélé meilleur que le jaune d'oeuf avec citrate ou lait pour la mobilité du sperme. Le jaune d'oeuf avec du lait fut meilleur que celui avec du citrate. Les spermes du taureau albinos et du taureau normal n'ont pas montré de différence significative dans leur mobilité. Resumen Tiempo de supervivencia de los espermatozoides normales de toro a 5 °C en diluentes a base de leche, citrato y tris-yema De un toro albino (pardo suizo) y un toro normal (holstein-frisón) se diluyeron cada vez 20 eyaculados con yema-citrato, leche-yema y tris (hidroximetilo) aminometano-yema, conservándose a 5 °C. En vista de la motilidad de los espermatozoides se mostró el diluente de tris-yema como mejor que el de leche-yema y que el de yema-citrato. Las motilidades de los espermatozoides del toro albino y del normal no presentaban diferencias significantes.

Prevalence of spermatozoa morphologic defects from Zebu bulls under free mating system

Bulls are keys to fertility and genetic improvement of the total herd. Spermatozoa quality is among of major components for evaluation of bulls to be used as breeding bulls. This study was therefore carried out to determine the prevalence of spermatozoa defects in Tanzania shorthorn breeding bulls, and to determine if the defects vary with scrotal circumference (SC), age and testicular pathology. Age was determined by dentition and SC measured by using standard scrotal metal. Sperm cells were collected by epididymal aspiration procedure and stained with Carbol-fuchsin. One hundred spermatozoa were counted per slide and each classified into normal or abnormal. Three testicular samples were taken after slaughter and processed for histological examination by staining with hemotoxylin and eosin. Forty sections of seminiferous tubules per section were evaluated and classified into either normal or abnormal. After examinations, 169 and 134 bulls were classified as satisfactory (normal) and unsatisfactory (abnormal) breeders, respectively. The mean SC (in centimeters) and percentages spermatozoa defects in normal and abnormal bulls were 28.9±2.6 and 15.6±2.5 versus 22.0±3.6 and 39.0±9.1, respectively. Most frequently observed primary spermatozoa defects in normal bulls were Stump-tail, DAG defect and decapitated sperm head while the secondary spermatozoa defects were loose head, bent tail, and proximal and distal cytoplasmic droplets. Bulls with severe testicular lesions had high percentages of spermatozoa defects which increased with severity of testicular abnormalities (P < 0.01). The results confirm the absence of proper criteria for selecting breeding bulls and support the earlier evidence of association between SC, testicular pathology and spermatozoa defects in bulls. Key words: Tanzania shorthorn zebu bull, spermatozoa defects, scrotal circumference, testicular pathology,

Cytogenetics of preimplantation embryos sired by bulls heterozygous for the 1/29 translocation.

Eighty-eight embryos aged 1 to 15 days sired by bulls heterozygous for the 1/29 chromosome translocation and 28 embryos aged 7 to 15 days sired by a normal bull were chromosomally analysed. All donor females had normal karyotypes. Only 47.4% of the embryos could be karyotyped. Two embryos sired by a heterozygous bull were trisomic. One 2-celled embryo was a 2n/ln mosaic and one 1-celled egg was polyspermic. Both of these were collected from superovulated donors. The mean cell number and mitotic index for 7-day embryos sired by heterozygous bulls were 72 and 3.73%, respectively, and sired by normal bulls, 81 and 5.74%, respectively.

Sexual functions, semen characteristics and fertility of bulls carrying the 1/29 chromosome translocation

Twelve bulls carrying the 1/29 chromosome translocation were compared with 45 chromosomally normal bulls concerning sexual functions, semen characteristics, and fertility. All bulls were unselected from the point of view of fertility. Sexual functions such as libido and serving ability were found to be quite normal in bulls carrying the 1/29 chromosome translocation. The semen analysis indicated small reductions in the concentration of semen and in the total number of spermatozoa but the volume of the ejaculate as well as qualitative characteristics of the semen such as wave motion and motility appeared normal. The fertility was reduced and the non-return rates to service at 28 and 56 days pointed to a reduction of 5 and 7%, respectively, expressed as percentage of the mean rates of the association.

Repression of Common Bull Sperm Flora andIn VitroImpairment of Sperm Motility withPseudomonas aeruginosaIntroduced by Contaminated Lubricant

Semen collected from clinically healthy bulls at an artificial insemination centre was examined for bacterial diversity. While bacteria that are normally present in the common flora of bovine semen were absent, such as Mycoplasma sp., Proteus sp. and Corynebacterium sp., all semen samples contained an unusually high number of Pseudomonas aeruginosa strains. Analysis via pulsed field gel electrophoresis demonstrated that one particular P. aeruginosa strain, present in a sealed bottle of lubricant, was widespread in bull semen. This strain was shown to secrete substances that inhibited both the growth of bacteria constituting the normal bull sperm flora and the motility of spermatozoa in vitro. This study demonstrated that commercially available lubricants might contain bacteria that can spread amongst breeding bulls and affect the quality of semen. Bacteriological controls and species' identification are necessary at several production levels, including lubricants and extenders, to ensure high semen quality and avoid the spread of pathogens.

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.

Meiotic recombination in normal and clone bulls and their offspring

Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny. The advent of somatic cell nuclear transfer (SCNT) as a reproductive biotechnology for use in livestock industry has made it easy to bypass these vital steps. However, few studies have been carried out on the impact of SCNT on the reproductive characteristics of cloned animals and, none to date, on the meiotic processes in animals, which were created by circumventing meiosis. In an attempt to assess the impact of cloning by SCNT on the meiotic processes, we undertook an immunocytological comparison of recombination in normal and clone bulls using antibodies raised against the synaptonemal complex protein 3 (SCP3) to label the lateral elements and the mismatch repair protein 1 (MLH1) foci on bivalents as indicators of recombination events. Our studies involving five normal bulls of proven fertility, two SCNT-derived bulls, and four mature offspring of SCNT bulls showed that the mean number of crossing over per spermatocyte for normal bulls (42 ± 4 SD; ranging from 33 to 56), was not significantly different from that of SCNT-derived bulls (43 ± 5 SD; ranging from 35 to 56), and the offspring of SCNT-derived bulls (43 ± 5 SD; ranging from 37 to 58). It would appear that circumventing meiosis to produce these animals does not influence the meiotic processes revealed by MLH1 foci detected in spermatocytes.

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

Multinuclear–Multiflagellar Sperm Defect in a Bull – a New Sterilizing Sperm Defect

The development and use of modern techniques, such as intracytoplasmic sperm injection (ICSI), gene knockout and sperm fluorescence in situ hybridization with chromosome- specific probes, have significantly increased our knowledge about sperm defects. We describe a new oligoasthenoteratozoospermic defect in a bull. Because of its morphological characteristics the defect was named the multinuclear-multiflagellar sperm defect. All spermatozoa in the ejaculate were abnormal. Many of the spermatozoa had multiple nuclei and multiple sperm tails. All spermatozoa lacked an acrosome, and only seldom did spermatozoa have a mitochondrial helix in the midpiece area. Meiosis and spermiogenesis were severely affected in this otherwise phenotypically normal bull. The sperm defects resembled the phenotype of a targeted gene knockout Hrb(-/-) (HIV-1 Rev-binding/interacting protein) mutant mouse strain, which is expressed as sterility in males, while females remain fertile. Since the father of this bull has been extensively used in at least three countries the defective gene has possibly become widespread in the red and white breeds (Ayrshire, Swedish Red and White, Norwegian Red) in the Nordic countries. However, it is not proved that the father of this bull is a carrier of this defect.

34 MEIOTIC RECOMBINATION IN SOMATIC CELL NUCLEAR TRANSFER BULLS AND THEIR OFFSPRING

In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) in the livestock industry, these vital steps have begun to be bypassed. Despite this, there have been few studies carried out on the reproductive characteristics of cloned animals and none to date regarding the consequences on the meiotic process. As these procedures grow in popularity and use, the importance of evaluating the long-term viability, health, and productivity of cloned animals and any subsequent offspring in future generations to validate potential applications of the technology is increasingly evident. Previously, studies of recombination and synapsis have focused on the physical observation of chiasmata formation in meiotic chromosomes; however, in recent years, the characterization of proteins that localize to the sites of crossing-over and of proteins present in the synaptonemal complex have permitted the study of meiotic recombination using a precise direct immunocytogenetic approach. Cytological analysis of meiotic cells obtained from the testicular tissue of five normal bulls of proven fertility, two SCNT transgenic bulls (Powell et al. 2004 Biol. Reprod. 71, 210–216, 2004), and four reproductively mature offspring of SCNT bulls was performed in order to detect the effects of SCNT on the meiotic process. Over 50 pachytene cells per animal were analyzed by immunofluorescence using antibodies against the synaptonemal complex protein 3 (SCP3) and the mismatch repair protein 1 (MLH1) located on the crossover sites. Data were analyzed using a mixed model analysis of variance with repeated measurements to determine group effects (SAS 9.1; SAS Institute, Inc., Cary, NC, USA). The average number of crossovers per spermatocyte for the non-SCNT bulls (42 � 4 SD, min: 33, max: 56), SCNT bulls (43 � 5 SD, min: 35, max: 56), and SCNT offspring (46 � 4 SD, min: 37, max: 58) were quite similar among the cells of the same individual; however, inter-individual variation was observed. These results are the first documentation of the normal range of variability of recombination distribution within the cattle genome and suggest that the SCNT process does not affect meiotic recombination. This work was funded by NSERC and the CRC program.