Identification of sperm proteins as biomarkers of field fertility in Holstein-Friesian bulls used for artificial insemination.

Despite passing stringent quality control, bulls used in artificial insemination can vary significantly in their fertility, emphasizing the need for reliable markers of sperm quality. This study aimed to identify sperm proteins acting as biomarkers of fertility in 2 different populations of dairy bulls classified based on their field fertility. Semen was collected and cryopreserved from: 54 Holstein bulls located in Ireland, classified according to fertility indexes as low fertility (LF, n = 23), medium fertility (n = 14), or high fertility (HF, n = 17); and 18 Holstein bulls located in Denmark, classified as LF (n = 8) or HF (n = 10). The proteome was measured through liquid chromatography-mass spectrometry and data were analyzed with the R software. Differentially abundant proteins between HF and LF bulls and biomarker proteins were determined through a modified t-test and random forest, respectively, selecting 301 differentially abundant proteins and 34 biomarker proteins. The predictive ability of the 34 biomarkers was evaluated employing support vector machine as the classifier, using their abundance levels in the Irish bulls to train the model and in the Danish bulls for validation. The prediction accuracy was 94.4%, with only one HF bull misclassified, corresponding to the lowest fertility index bull in the HF group. The biomarkers more abundant in sperm of HF bulls enriched axoneme assembly and sperm motility (false discovery rate <0.05), according to functional analysis. In conclusion, a robust model coupled with the application of appropriate bioinformatic tools allowed the identification of functionally relevant sperm proteins predictive of the fertility of Holstein bulls used in artificial insemination.

Influence of sire fertility status on conceptus-induced transcriptomic response of the bovine endometrium.

The aim was to examine the effect of sire fertility status on conceptus-induced changes in the bovine endometrial transcriptome. To generate elongated conceptuses, Day 7 blastocysts produced in vitro using frozen-thawed sperm from Holstein Friesian bulls (3 High fertility, HF and 3 Low fertility, LF) were transferred in groups of 5-10 into synchronized heifers (n = 7 heifers per bull) and recovered following slaughter on Day 15. Day 15 endometrial explants recovered from the uterine horn ipsilateral to the corpus luteum were recovered from synchronized cyclic heifers (n = 4). Explants from each heifer were co-cultured for 6 h in RPMI medium alone (Control) or with 100 ng/ml ovine recombinant interferon tau (IFNT) or with a single conceptus from each HF or LF bull. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq and the resulting data were analyzed with R software. The numbers of differentially expressed genes (DEG; FDR<0.05) were: HF vs. Control: 956; LF vs. Control: 1021; IFNT vs. Control: 1301; HF vs. LF: 2. Unsurprisingly, the majority of DEG (658) were common to all comparisons and were related to IFNT-induced changes in the endometrium. Prior to applying the adjusted p-value, there were 700 DEG between HF and LF, with 191 and 509 genes more expressed in HF or LF, respectively (p < 0.05). Overrepresentation analysis of KEGG pathways (FDR<0.05), revealed that DEG with higher expression in LF were involved in cell cycle and proteolysis, while those upregulated DEG by HF conceptuses were strongly associated with immune process pathways, such as TNF, NF-kappa B, cytokine-cytokine receptor interaction, and TLR signaling. These pathways were also enriched by DEG upregulated by IFNT compared to the Control. Furthermore, only the HF, and not the LF group, affected the expression of most genes in these pathways (p < 0.05) according to a negative binomial regression model. Finally, a weighted gene co-expression network analysis revealed two clusters of co-expressed genes associated with the HF conceptuses (p < 0.05), which were also enriched for the aforementioned pathways. In conclusion, HF conceptuses, similar to IFNT treatment, stimulated multiple pathways involved in immune response, which were apparently not affected by LF conceptuses.

Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility.

Younger bulls typically produce lower volumes of semen per ejaculate with a lower sperm concentration than older more mature, bulls and often fail to meet semen demand using standard collection frequency schedules. The objective of this study was to assess the effect of ejaculate collection frequency on semen output, sperm quality and field fertility in young bulls under commercial conditions. Holstein Friesian bulls aged 366 ± 8 days (mean ± SEM) were assigned one of two ejaculate collection frequencies: (i) HF (n = 14 bulls), where ejaculates were collected twice a day, five days in each two-week period or (ii) LF (n = 12 bulls), where ejaculates were collected once a day, two days per week. The trial period continued until each bull reached both 20 ejaculates and 1000 marketable frozen semen straws. Subjective motility was assessed on all ejaculates pre-freeze and post-thaw (at 0 and 2 h). A subset of ejaculates were assessed post-thaw by computer-assisted sperm analysis for motility, kinematics and morphological defects and by flow cytometry for viability, membrane fluidity, acrosome integrity, reactive oxygen species and DNA fragmentation. A total of 13,846 inseminations (9,541 for HF and 4,305 for LF) were carried out on dairy cows and heifers. HF reached the 1000 straw threshold 41 days earlier than LF (P < 0.01) with the same number of ejaculates. Ejaculate volume and sperm concentration were not affected by treatment but the first ejaculate of the day (HF only) had a greater volume (P < 0.001) and sperm concentration (P < 0.05) than the second ejaculate. HF had higher pre-freeze total (P < 0.01) and gross (P < 0.05) motility than LF. HF had higher post-thaw (2 h) total and gross motility than LF (P < 0.05). Ejaculate rejection rates did not differ between treatments. There was no effect of treatment, week or ejaculate number of the day (HF only) on post-thaw motility and kinematic parameters or sperm viability, membrane fluidity, acrosome integrity and DNA fragmentation. However, HF had lower superoxide production than LF (P < 0.05). Pregnancy per artificial insemination was 64.5 ± 1.0% and 59.9 ± 1.1% for the HF and LF bulls, respectively (mean ± SEM; P = 0.05). In conclusion, collecting ejaculates more frequently from young bulls significantly reduced the number of days required to obtain 1000 straws, increased semen quality in terms of lower superoxide production and increased field fertility.

An ex-vivo assessment of differential sperm transport in the female reproductive tract between high and low fertility bulls.

Despite passing all quality control checks at animal breeding centres, bulls with apparently normal semen quality can yield unacceptably low field fertility rates. This study took an ex-vivo approach to assess if bulls of divergent field fertility differ in the ability of their spermatozoa to interact with the female reproductive tract and its secretions. Six high and six low fertility Holstein Friesian bulls (+4.0 ± 0.2 and -15.7 ± 3.13, respectively; adjusted mean fertility ± s.e.m. mean of the bull population was 0) were selected from a population of 840 bulls with >500 field inseminations per bull. Thawed spermatozoa from each bull were analysed across a range of in vitro assays to assess their ability to transverse the female reproductive tract including; motility and kinematic parameters using computer-assisted sperm analysis, viability, membrane fluidity and acrosomal integrity using flow cytometry as well as mucus penetration tests, rheotactic behaviour and sperm binding ability to the oviductal epithelium. While there was no significant difference between high and low fertility bulls in most of the sperm motility, kinematic and sperm functional parameters (namely, motility, average path velocity, linearity, straightness, amplitude of lateral head movement), viability, membrane fluidity or acrosome intactness, high fertility bulls had higher curvilinear velocity compared to the low fertility group (P < 0.05) and a higher straight-line velocity was observed although it did not reach statistical significance (P = 0.08). There was no difference between treatment groups in the ability of spermatozoa to penetrate periovulatory cervical mucus or in their rheotactic response (P > 0.05). Interestingly, there was a positive correlation between the straight-line velocity of spermatozoa and their rheotactic response (r = 0.45, P < 0.001) and further linear regression analysis indicated 18.9% of the variance in sperm rheotaxis was accounted for by straight line velocity. A higher number of spermatozoa from the high fertility group compared to the low fertility group bound to oviductal explants (15.1 ±â€¯0.98 and 12.5 ±â€¯0.76, respectively; mean ±â€¯s.e.m; P < 0.05). In conclusion, the differences in the kinematics of sperm motility and ability to bind to oviductal explants between high and low fertility bulls were modest and are unlikely to explain the inherent differences in fertility between these cohorts of bulls.

Heat illness experience at BMH Shaibah, Basra, during Operation TELIC: May-July 2003.

This is an observational study of heat-related illness in UK Service Personnel deployed into summer conditions in Northern Kuwait and Southern Iraq. Among 622 hospitalisations reported during a 9-week period at the historical British Military Hospital, Shaibah, 303 consecutive admissions are reviewed in detail. Several clinical syndromes attributable to thermal stress were observed. These ranged from self-limiting debility to life-threatening failures of homeostasis, with 5.0% developing a critical care requirement. Hyponatraemia was a commonly occurring electrolyte disturbance by which, relative to the local reference range, a majority of heat-attributed admissions were affected. Reductions in measured serum sodium could be profound (<125 mmol/L in 20.1% of all heat-related casualties). Hypokalaemia was observed in half of cases, though only a minority were affected by severely low potassium (<2.5 mmol/L in 4.0%). Despite preventive measures prescribed on hospital discharge, illness and significant biochemical derangements could recur upon return to duties in the heat. We reiterate the need for primary prevention of heat illness wherever possible and importance of early, effective interventions to treat and protect Service Personnel from secondary injury. We also highlight the requirement for comprehensive assessment to inform prognostication and occupational decision-making in relation to extreme climatic heat, including aeromedical evacuation. We draw additional attention to the contribution of psychological factors in select cases and identify research questions to improve understanding of environment-induced incapacitation in general.

Comparison of the uterine inflammatory response to frozen-thawed sperm from high and low fertility bulls.

Some bulls with apparently normal semen quality yield unacceptably low pregnancy rates. We hypothesised that a differential uterine immunological response to sperm from high and low fertility bulls may contribute to these differences. The experimental model used was heifer follicular phase uterine explants incubated with frozen-thawed sperm from high and low fertility bulls (3-5 replicates per experiment). Inflammatory gene expression of IL1A, IL1B, IL6, TNFA and CXCL8 were assessed by qPCR and IL1-ß and IL-8 were quantified in explant supernatants by ELISA. Neutrophil binding affinity to sperm from high and low fertility bulls was also assessed. There was a significant up-regulation of IL1A, IL1B and TNFA from frozen-thawed sperm, irrespective of fertility status, compared to the unstimulated control. This response was confirmed at the protein level, with an increase of IL-1ß and IL-8 protein concentrations by 5 and 2.7 fold, respectively (P < 0.05). Although no significant differences in the inflammatory response at the gene or protein level were evident between high and low fertility bulls, more sperm from low compared to high fertility bulls bound to neutrophils (P < 0.05). Using bulls of unknown fertility, cauda epididymal sperm (CES) plus seminal plasma (SP) upregulated IL6 (P < 0.05) but there was no upregulation of any inflammatory gene expression for CES alone. Overall, this ex vivo study demonstrated an upregulation of inflammatory gene expression in the uterus in response to frozen-thawed bull sperm. While there was no difference between sperm from high and low fertility bulls, there was a greater binding affinity of low fertility sperm by neutrophils.

Sire contribution to fertilization failure and early embryo survival in cattle.

Despite passing routine laboratory tests of semen quality, bulls used in artificial insemination (AI) exhibit a significant range in field fertility. The objective of this study was to determine whether subfertility in AI bulls is due to issues of sperm transport to the site of fertilization, fertilization failure, or failure of early embryo or conceptus development. In experiment 1, Holstein-Friesian bulls (3 high fertility, HF, and 3 low fertility, LF) were selected from the national population of AI bulls based on adjusted fertility scores from a minimum of 500 inseminations (HF: +4.37% and LF: -12.7%; mean = 0%). Superovulated beef heifers were blocked based on estimated number of follicles at the time of AI and inseminated with semen from HF or LF bulls (n = 3-4 heifers per bull; total 19 heifers). Following slaughter 7 d later, the number of corpora lutea was counted and the uteri were flushed. Recovered structures (oocytes/embryos) were classified according to developmental stage and stained with 4',6-diamidino-2-phenylindole to assess number of cells and accessory sperm. Overall recovery rate (total structures recovered/total corpora lutea) was 52.6% and was not different between groups. Mean (± standard error of the mean) number of embryos recovered per recipient was 8.7 ± 5.2 and 9.4 ± 5.5 for HF and LF, respectively. Overall fertilization rate of recovered structures was not different between groups. However, more embryos were at advanced stages of development (all blastocyst stages combined), reflected in a greater mean embryo cell number on d 7 for HF versus LF bulls. Number of accessory sperm was greater for embryos derived from HF than for LF bulls. The aim of experiment 2 was to evaluate the effect of sire fertility on survival of bovine embryos to d 15. Day 7 blastocysts were produced in vitro using semen from the same HF (n = 3) and LF (n = 3) bulls and transferred in groups of 5-10 to synchronized heifers (n = 7 heifers per bull; total 42 heifers). Conceptus recovery rate on d 15 was higher in HF (59.4%,) versus LF (45.0%). Mean length of recovered conceptuses for HF bulls was not affected by fertility status. In conclusion, while differences in field fertility among AI sires used in this study were not reflected in fertilization rate, differences in embryo quality were apparent as early as d 7. These differences likely contributed to the higher proportion of conceptuses surviving to d 15 in HF bulls.

Ewe breed differences in cervical anatomy and cervicovaginal mucus properties: An international study.

In sheep, cervical artificial insemination (AI) involves depositing semen at the cervical opening, as it is not possible to traverse the cervix due to its complex anatomy. However, internationally this method yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway, in which vaginal deposition of frozen-thawed semen to a natural estrus yields pregnancy rates around 70%. As the cervix and its secretions are the principal factors influencing sperm transport to the site of fertilization the aim of this study was to characterise the differences in the cervical anatomy as well as the cervicovaginal mucus properties of six European ewe breeds across three countries known to have differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk and Belclare in Ireland, Fur and Norwegian White Sheep (NWS) in Norway and Ile de France and Romanov in France (n = 28-30 ewes/breed). Cervicovaginal mucus was collected at the follicular and luteal phases of both a synchronized and natural cycle and assessed for mucus weight, viscosity and colour. The anatomical characteristics of the cervix (length of the cervix, number of cervical rings and the appearance of the external os) were assessed post-mortem. There was a type of the cycle by ewe breed interaction represented by no differences in mucus production between ewe breeds at the natural cycle for both the follicular and luteal phases of the cycle. However, there were differences between ewe breeds at the synchronized cycle (P < 0.05). Belclare had the lowest mucus production at the follicular phase while NWS had the lowest amount of mucus at the luteal phase of the synchronized cycle. Overall, across all ewe breeds, mucus production was higher at the follicular than at the luteal phase (P < 0.05). Despite reports of Suffolk and NWS having the most divergent pregnancy rates following cervical AI with frozen-thawed semen, both breeds had the lowest overall mucus viscosity at the follicular phase of both types of cycle with no differences between both ewe breeds (P > 0.05). The length of the cervix, number of cervical rings and the external os type were affected by ewe breed (P < 0.05). Suffolk ewes had longer cervices but lower number of cervical rings than NWS and Fur ewes (both with higher pregnancy rates). In conclusion, while mucus production and mucus viscosity was affected by breed, these changes are not consistent with the known differences between ewe breeds in their pregnancy rates following cervical AI with frozen-thawed semen.

Population structure and breed composition prediction in a multi-breed sheep population using genome-wide single nucleotide polymorphism genotypes.

Knowledge of population structure and breed composition of a population can be advantageous for a number of reasons; these include designing optimal (cross)breeding strategies in order to maximise non-additive genetic effects, maintaining flockbook integrity by authenticating animals being registered and as a quality control measure in the genotyping process. The objectives of the present study were to 1) describe the population structure of 24 sheep breeds, 2) quantify the breed composition of both flockbook-recorded and crossbred animals using single nucleotide polymorphism BLUP (SNP-BLUP), and 3) quantify the accuracy of breed composition prediction from low-density genotype panels containing between 2000 and 6000 SNPs. In total, 9334 autosomal SNPs on 11 144 flockbook-recorded animals and 1172 crossbred animals were used. The population structure of all breeds was characterised by principal component analysis (PCA) as well as the pairwise breed fixation index (Fst). The total number of animals, all of which were purebred, included in the calibration population for SNP-BLUP was 2579 with the number of animals per breed ranging from 9 to 500. The remaining 9559 flockbook-recorded animals, composite breeds and crossbred animals represented the test population; three breeds were excluded from breed composition prediction. The breed composition predicted using SNP-BLUP with 9334 SNPs was considered the gold standard prediction. The pairwise breed Fst ranged from 0.040 (between the Irish Blackface and Scottish Blackface) to 0.282 (between the Border Leicester and Suffolk). Principal component analysis revealed that the Suffolk from Ireland and the Suffolk from New Zealand formed distinct, non-overlapping clusters. In contrast, the Texel from Ireland and that from New Zealand formed integrated, overlapping clusters. Composite animals such as the Belclare clustered close to its founder breeds (i.e., Finn, Galway, Lleyn and Texel). When all 9334 SNPs were used to predict breed composition, an animal that had a majority breed proportion predicted to be ≥0.90 was defined as purebred for the present study. As the panel density decreased, the predicted breed proportion threshold, used to identify animals as purebred, also decreased (≥0.85 with 6000 SNPs to ≥0.60 with 2000 SNPs). In all, results from the study suggest that breed composition for purebred and crossbred animals can be determined with SNP-BLUP using ≥5000 SNPs.

Implications of boar sperm kinematics and rheotaxis for fertility after preservation.

Artificial insemination (AI) is the single most important assisted reproductive technique devised to facilitate the genetic improvement of livestock. In the swine industry, it has broadly replaced natural service over the last number of decades which has been made possible by the high pregnancy rates and litter sizes obtainable with semen extended, up to, and sometimes beyond 5 d. Central to achieving good reproductive performance is the ability of boar studs to monitor semen quality, the basis of which has long been the assessment of sperm motility by subjective and, more recently, by more objective computerised systems. In this review, the literature on the relationship between sperm motility and kinematic parameters and field fertility is summarised. We discuss how this relationship is dependent on factors such as the viscosity of the media and the use of standard operating procedures. Emerging evidence is discussed regarding the importance of sperm rheotaxis and thigmotaxis as long-distance sperm guidance mechanisms, which enable motile functional spermatozoa to avoid the backflow of fluid, mucus and semen from the sow's uterus in the hours post AI, facilitating the establishment of sperm reservoirs in the oviducts. The literature on the use of microfluidics in studying sperm rheotaxis in vitro is also summarised, and we discuss how these systems, when combined with techniques such as lensless microscopy, have the potential to offer more physiological assessments of the swimming patterns of boar spermatozoa. Finally, possible future avenues of further investigation are proposed.

Progesterone induces the release of bull spermatozoa from oviductal epithelial cells.

The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose-response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca2+ channel antagonist; 5µM), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2µM), mifepristone (a classical and membrane P4 receptor antagonist; 400nM) and AG205 (a membrane P4 receptor antagonist; 10µM). BOEC explants were incubated with frozen-thawed bovine spermatozoa for 30min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30min with either no treatment, P4, NNC, mibefradil, P4+mibefradil, P4+NNC, P4+mibefradil+NNC or P4+EGTA. In Experiment 2, BOECs were treated for 30min with either no treatment, P4, mifepristone, AG205, mifepristone+AG205, P4+mifepristone, P4+AG205 or P4+mifepristone+AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca2+.

The biological mechanisms regulating sperm selection by the ovine cervix.

In species where semen is deposited in the vagina, the cervix has the unique function of facilitating progress of spermatozoa towards the site of fertilisation while also preventing the ascending influx of pathogens from the vagina. For the majority of species, advances in assisted reproduction techniques facilitate the bypassing of the cervix and therefore its effect on the transit of processed spermatozoa has been largely overlooked. The exception is in sheep, as it is currently not possible to traverse the ovine cervix with an inseminating catheter due to its complex anatomy, and semen must be deposited at the external cervical os. This results in unacceptably low pregnancy rates when frozen-thawed or liquid stored (>24 h) semen is inseminated. The objective of this review is to discuss the biological mechanisms which regulate cervical sperm selection. We assess the effects of endogenous and exogenous hormones on cervical mucus composition and discuss how increased mucus production and flow during oestrus stimulates sperm rheotaxis along the crypts and folds of the cervix. Emerging results shedding light on the sperm-cervical mucus interaction as well as the dialogue between spermatozoa and the innate immune system are outlined. Finally, ewe breed differences in cervical function and the impact of semen processing on the success of fertilisation, as well as the most fruitful avenues of further investigation in this area are proposed.

Cervical mucus sialic acid content determines the ability of frozen-thawed ram sperm to migrate through the cervix.

The aim of this study was to investigate the properties and to functionally characterize the cervical mucus that modulates sperm transport through the cervix by using ewe breeds with a divergent pregnancy rate (Belclare and Suffolk; high and low, respectively) following cervical insemination using frozen-thawed semen. Sperm number, as well as sialic acid and fucose content in both the channels and in the lumen of different regions of the cervix were quantified in inseminated Belclare and Suffolk ewes. Expression of glycosyltransferase and MUC genes, glycosidase activity and sialic acid speciation in follicular phase cervical tissue and mucus were assessed. More spermatozoa were found in the cervical channels in the region closest to the cervical os in Belclare than Suffolk ewes (P < 0.05) and Suffolk ewes had a higher sialic acid content in the cervical channels than Belclare ewes (P < 0.05) in all regions of cervix. Suffolk ewes had significantly higher expression of FUT1, ST6GAL1 and MUC5AC than Belclare ewes. There was no difference between the breeds in glycosidase activity (P > 0.05). Levels of Neu5Ac were higher in Belclare than Suffolk ewes (P < 0.05) and levels of Neu5Gc was higher in Suffolk than Belclare ewes (P < 0.05). Competitive sperm penetration assays demonstrated that frozen-thawed sperm progression increased when cervical mucus was incubated with sialyllactose prior to a sperm penetration test (P < 0.05). These results suggest that the difference between Belclare and Suffolk ewes in sperm transport with frozen-thawed semen is due to the higher concentration of sialic acid within channels, which binds to spermatozoa and reduces their ability to traverse the cervix.

Effect of Early Calf-Hood Nutrition on the Transcriptional Regulation of the Hypothalamic-Pituitary-Testicular axis in Holstein-Friesian Bull Calves.

The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of the arcuate nucleus of the hypothalamus, anterior pituitary and testes in Holstein-Friesian bulls. Holstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were offered a high (n = 10) or low (n = 10) plane of nutrition in order to achieve an overall growth rate of 1.2 and 0.5 kg/day. At 126 (±3) days of age, calves were euthanized, hypothalamus (arcuate region), anterior pituitary and testicular parenchyma samples were harvested and RNAseq analysis was performed. There were 0, 49 and 1,346 genes differentially expressed in the arcuate nucleus, anterior pituitary and testicular tissue of bull calves on the low relative to the high plane of nutrition, respectively (P < 0.05; False Discovery Rate <0.05). Cell cycle processes in the anterior pituitary were down regulated in the low relative to the high plane of nutrition; there was no differential expression of genes related to reproductive processes. Gene expression involved in cholesterol and androgen biosynthesis in the testes were down regulated in animals on the low plane of nutrition. This study provides insight into the effect of early life plane of nutrition on the regulation of the HPT axis.

Role of early life nutrition on regulating the hypothalamic­anterior pituitary­testicular axis of the bull

The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein­Friesian bull calves. Holstein­Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a HIGH (n = 10) or LOW (n = 10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 ± 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein­Friesian bulls (n = 10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P< 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P < 0.001), had larger testes (P < 0.001), larger seminiferous tubule diameter (P < 0.001), more mature spermatogenic cells (P < 0.001) and more Sertoli cells (P < 0.05) in accordance with both morphological and transcriptional data. Overall, testicular gene expression profiles suggested a more mature stage of development in HIGH compared with LOW and were more closely aligned to that of mature bulls. Ghrelin receptor was the only differentially expressed gene between LOW and HIGH calves in either the anterior pituitary (P < 0.05) or arcuate nucleus region of the hypothalamus (P < 0.10) and was upregulated in LOW for both tissues. This study indicates that an enhanced plane of nutrition during early calfhood favourably alters the biochemical regulation of the hypothalamus­anterior pituitary­testicular axis, advancing testicular development and hastening spermatogenesis.

Review: Understanding the causes of variation in reproductive wastage among bulls.

The ability to predict the fertility of bulls before semen is released into the field has been a long-term objective of the animal breeding industry. However, the recent shift in the dairy industry towards the intensive use of young genomically selected bulls has increased its urgency. Such bulls, which are often in the highest demand, are frequently only used intensively for one season and consequently there is limited time to track their field fertility. A more pressing issue is that they produce fewer sperm per ejaculate than mature bulls and therefore there is a need to reduce the sperm number per straw to the minimum required without a concomitant reduction in fertility. However, as individual bulls vary in the minimum number of sperm required to achieve their maximum fertility, this cannot be currently achieved without extensive field-testing. Although an in vitro semen quality test, or combination of tests, which can accurately and consistently determine a bull's fertility and the optimum sperm number required represent the 'holy grail' in terms of semen assessment, this has not been achieved to date. Understanding the underlying causes of variation in bull fertility is a key prerequisite to achieving this goal. In this review, we consider the reliability of sire conception rate estimates and then consider where along the pregnancy establishment axis the variation in reproductive loss between bulls occurs. We discuss the aetiology of these deficiencies in sperm function and propose avenues for future investigation.

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

Primate ß-defensin 126 regulates the ability of spermatozoa to bind to oviductal epithelial cells invitro. Bovine ß-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 invivo, 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.

Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro.

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus-oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm-oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.