The role of β-defensin 126 on the ability of bull sperm to bind oviductal epithelium

Previous studies in primates have shown that β-defensin 126 regulates the ability of sperm to bind to oviductal epithelial cells, whilst in humans; mutations in the β-defensin 126 gene have been linked to male subfertility. Bovine β-defensin 126 (BBD126) exhibits preferential expression for the caudal epididymis of the bull but there have been no studies on its functional role in cattle. The aim of this study was to examine the role of BBD126 and BBD126 genetic variation on sperm motility, bovine oviductal epithelial cell (BOEC) binding ability and on sperm agglutination. In a previous study carried out by a member of our research group, Dr Emma Finlay, adjusted bull fertility phenotypes (based on a minimum of 1000 inseminations) for 7000 AI bulls were used to identify bulls of high and low fertility. The most divergent bulls (n=150) were selected for targeted sequencing of β-defensin genes and an association study was performed to identify genetic regions associated with sire conception rate. The most significantly associated single nucleotide polymorphisms (SNPs) were located in a haplotype consisting of 94 SNPs over 138kb, which included the β-defensin 126 gene, found only in bulls of high fertility. In this study, to examine the effect of the haplotype on sperm function, frozen-thawed sperm from high fertility bulls with (H+ive; n=4) and without (H-ive; n=4) the haplotype as well low fertility bulls without the haplotype (L-ive; n=4) were assessed for post-thaw motility using computer aided sperm analysis (CASA) and assessed for binding ability using in vitro BOEC binding assays (both monolayers and explants). BBD126 haplotype was found to have no effect on post-thaw sperm motility, however, bulls of high fertility with the haplotype (H+ive) had an increased ability to bind BOEC explants in comparison to bulls of high (H-ive) and low fertility (L-ive) without the haplotype (P<0.05). BBD126 has been shown to be highly resistant to methods of dissociation used in other species and, as a result, corpus epididymis sperm, a model in which the protein is not present, was used to study the functional role of BBD126 in sperm binding and agglutination. Corpus sperm were incubated with recombinant BBD126 (rBBD126) in the absence or presence of BBD126 antibody. Addition of rBBD126 enhanced the ability of sperm to bind BOEC and reduced sperm agglutination (P<0.05). The presence of the antibody inhibited the increase in sperm binding ability, however, it failed to abrogate the effect of the protein on sperm agglutination. These findings indicate that BBD126 and BBD126 haplotype plays a role in bovine sperm binding and that BBD126 protein has a non-specific effect on sperm agglutination

The in vitro addition of docosahexaenoic acid (DHA) improves the quality of cooled but not frozen-thawed stallion semen

The aim of this study was to assess the effect of the addition of docosahexanoic acid (DHA) on the in vitro quality of cooled and frozen-thawed stallion semen. In Experiment 1, semen from 10 stallions was collected (3 ejaculates per stallion). Semen was diluted to 100 x 106 spermatozoa/mL with 0.02 mM of vitamin E (VE) and 0, 1, 10 or 20 ng of DHA/mL and frozen. Semen was thawed and total motility (TM), acrosome integrity and morphology were assessed. In Experiment 2, semen from 3 stallions was collected (3 ejaculates per stallion) and frozen as in Experiment 1, but VE and DHA were added after thawing. Total motility and progressive linear motility (PLM) were assessed at 30, 60 and 120 min and viability, acrosome integrity and membrane fluidity at 30 min. In Experiment 3, semen from 5 stallions was collected (1-3 ejaculates per stallion), diluted to 20 x 106 spermatozoa/mL and stored at 4ºC. After 1, 24, 48 and 72 h, TM, PLM, viability, membrane fluidity and lipid peroxidation were assessed. The addition of DHA had no effect on frozen semen (Experiments 1 and 2) but improved TM, PLM and membrane fluidity in cooled stallion semen

Mean vigorous physical activity across tertiles of cardiorespiratory fitness.

<p>*Significantly different to “Middle” fitness group, p<0.05.</p

Mean BMI grade across tertiles of cardiorespiratory fitness.

<p>**Significantly different to “Middle” and “High” fitness group, p<0.01. *Significantly different to “”High” fitness groups, p<0.05.</p

Relationship between cardiorespiratory fitness, systolic blood pressure, body mass index, waist circumference and waist-height ratio.

<p>^aadjusted for age, sex and GMFCS level</p><p>^badjusted for age, sex, GMFCS level and BMI grade; zSBP, z-scores for systolic blood pressure</p><p>Relationship between cardiorespiratory fitness, systolic blood pressure, body mass index, waist circumference and waist-height ratio.</p

Mean waist-height ratio across tertiles of cardiorespiratory fitness.

<p>*Significantly different to “Middle” and “High” fitness groups, p<0.001.</p

Characteristics of participants (n = 55).

<p>zSBP, z-scores for systolic blood pressure; zDBP, z-scores for diastolic blood pressure; GMFCS, Gross Motor Function Classification System</p><p>Characteristics of participants (n = 55).</p

Patterned Enzymatic Degradation of Poly(ε-caprolactone) by High-Affinity Microcontact Printing and Polymer Pen Lithography

This paper reports deposition of Candida antarctica Lipase B (CALB) on relatively thick poly­(ε-caprolactone) (PCL) films (300–500 nm) to create well-defined patterns using two different writing techniques: high-affinity microcontact (HA-μCL) and polymer pen (PPL) lithography. For both, an aqueous CALB ink is absorbed onto a polydimethylsiloxane (PDMS) writing implement (PDMS stamp or a PDMS pen tip), which is transferred to a spun-cast PCL film. HA-μCL experiments demonstrated the importance of applied pressure to obtain high-resolution patterns since uniform contact is needed between raised 20 μm parallel line regions of the PDMS stamp and the surface. AFM imaging shows pattern formation evolves gradually over incubation time only in areas stamped with CALB cutting through spherulites without apparent influence by grain boundaries. Strong binding of CALB to PCL is postulated as the mechanism by which lateral diffusion is limited. PPL enables formation of an arbitrary image by appropriate programming of the robot. The PDMS pen tips were coated with an aqueous CALB solution and then brought into contact with the PCL film to transfer CALB onto the surface. By repeating the ink transfer step multiple times where pen tips are brought into contact with the PCL film at a different locations, a pattern of dots is formed. After printing, patterns were developed at 37 °C and 95% RH. Over a 7-day period, CALB progressively etched the PCL down to the silicon wafer on which it was spun (350 nm) giving round holes with diameters about 10 μm. AFM images show the formation of steep PCL walls indicating CALB degraded the PCL film in areas to which it was applied. This work demonstrates that high-resolution patterns can be achieved without immobilizing the enzyme on the surface of polymeric stamps that limits the depth of features obtained as well as the throughput of the process

Recombinant β-defensin 126 promotes bull sperm binding to bovine oviductal epithelia

Primate β-defensin 126 regulates the ability of spermatozoa to bind to oviductal epithelial cells in vitro. Bovine β-defensin 126 (BBD126) exhibits preferential expression in the cauda epididymis of the bull, but there have been few studies on its functional role in cattle. The aim of the present study was to examine the role of BBD126 in bull sperm binding to bovine oviductal epithelial cell (BOEC) explants. BBD126 has been shown to be highly resistant to the standard methods of dissociation used in other species and, as a result, corpus epididymal spermatozoa, which have not been exposed to the protein, were used to study the functional role of BBD126. Corpus epididymal spermatozoa were incubated with recombinant (r) BBD126 in the absence or presence of anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of epididymal spermatozoa to bind to BOEC explants (P < 0.05). Anti-BBD126 antibody blocked the BBD126-mediated increase in sperm binding capacity. Ejaculated spermatozoa, which are coated with native BBD126 protein but also a large number of seminal plasma proteins in vivo, were incubated with rBBD126 in the absence or presence of the anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of ejaculated spermatozoa to bind to BOEC explants (P < 0.05), whereas rBBD126 also reduced corpus sperm agglutination (P < 0.05). These results suggest that, similar to the role of its analogue in the macaque, spermatozoa with more BBD126 in their acrosome may represent spermatozoa with more oviduct binding capacity

A dual targeted β-defensin and exome sequencing approach to identify, validate and functionally characterise genes associated with bull fertility

Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertilityrelated β-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple β-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine β-defensin genes and functional analysis supports a role for β-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle