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K Bender, S Walsh, A C O Evans, T Fair and L Brennan

There has been a marked decline in the fertility of dairy cows over the past decades, and metabolomic analysis offers a potential to investigate the underlying causes. Metabolite composition of the follicular fluid, which presents the intrafollicular environment, may be an important factor affecting oocyte maturation and subsequent early embryo development. The aim of the present study was to investigate the metabolic differences between follicular fluid from the dominant follicle of lactating cows and heifers using gas chromatography mass spectrometry (GC–MS)-based metabolomics. Follicular fluid and serum were collected from cows and heifers over three phases of follicle development: newly selected dominant follicles, preovulatory follicles prior to oestrus and post-LH surge follicles. Analysis of the fatty acids revealed that there were 24 fatty acids and 9 aqueous metabolites significantly different between cows and heifers. Of particular interest were the higher concentrations of saturated fatty acids (palmitic acid, P=0.001; stearic acid, P=0.005) in follicular fluid from cows and higher docosahexaenoic acid levels (P=0.022) in follicular fluid from heifers. Analysis of the metabolite composition of serum revealed that follicular fluid had a unique lipid composition. The higher concentrations of detrimental saturated fatty in cows will have a negative impact on oocyte maturation and early embryo development. Overall, the results suggest that the follicle microenvironment in cows potentially places their oocytes at a developmental disadvantage compared with heifers, and that this may contribute to well-characterised differences in fertility.

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S. Fair, Kieran Meade, Karine Reynaud, Xavier Druart and Simon Paul de Graaf

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 it’s 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 (>24h) 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.

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B Fernandez-Fuertes, A Blanco-Fernandez, C J Reid, K G Meade, S Fair and P Lonergan

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.

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L Richardson, J P Hanrahan, T Tharmalingam, S D Carrington, P Lonergan, A C O Evans and S Fair

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.

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A M English, D A Kenny, C J Byrne, H Sauerwein, C Urh, M A Crowe, C Staub, S M Waters and S Fair

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 Holstein–Friesian bull calves. 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 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 Holstein–Friesian 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 hypothalamus–anterior pituitary–testicular axis, advancing testicular development and hastening spermatogenesis.

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D Corcoran, T Fair, S Park, D Rizos, O V Patel, G W Smith, P M Coussens, J J Ireland, M P Boland, A C O Evans and P Lonergan

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.