Peter M. Hastie

hCG treatment on day of mating improves embryo viability and fertility in ewe lambs

An investigation was conducted to determine the effects of supplementing hCG at mating time on luteal function, conceptus growth, placentation and reproductive performance in TexelxClun Forest ewe lambs. After oestrus synchronisation with progestagen sponges and PMSG ewe lambs were treated either with normal saline (n=102) or 150 IU of hCG (n=105) at mating time. At 25 days after mating 24 animals were slaughtered from each group to determine embryo viability and placental development. hCG significantly (P<0.05) increased crown-rump length (saline: 11.9+/-0.2 mm; hCG: 12.7+/-0.2 mm), amniotic sac width (saline: 11.4+/-0.4 mm; hCG: 12.0+/-0.3 mm) and the number of placentomes (saline: 90.8+/-7.3; hCG=122.4+/-6.3). Among the pregnant animals that were slaughtered on 25 days post-mating, ovulation rate tended to be higher in the hCG group compared to controls (saline: 1.16; hCG: 1.54). However, no difference was observed either in mean plasma progesterone concentrations (saline: 4.6 ng/ml; hCG: 4.9 ng/ml; sed 0.56) or in progesterone production from luteal slices when cultured in vitro (saline: 239.6+/-11.8 ng/mg; hCG: 263.2+/-13.6 ng/mg) between controls and hCG treated animals. Reproductive performance was also recorded in ewe lambs that were either treated with saline (n=78) or hCG (n=81). The total number of lambs born (saline: 38; hCG: 58) was significantly (P<0.05) greater in hCG group compared to saline-treated controls. Both lambing percentage (saline: 36%; hCG: 48%) and litter size (saline: 1.35; hCG: 1.48) tended to be greater (P<0.10) in hCG-treated animals compared to the controls. In conclusion, these data suggest that treatment of ewe lambs with hCG at the time of mating improves conceptus growth, placentation and number of lambs born.

Semi-quantitative analysis of Ruminococcus flavefaciens, Fibrobacter succinogenes and Streptococcus bovis in the equine large intestine using real-time polymerase chain reaction.

There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA sequences for specific detection of candidate bacterial species. Overall, in frozen and lyophilised digesta, there were significantly (P F. succinogenes > S. bovis (P < 0.05), while in lyophilised digesta R. flavefaciens was present in significantly (P < 0.05) greater amounts than F. succinogenes and S. bovis. R. flavefaciens and F. succinogenes were abundant at significantly (P < 0.05) greater levels in lyophilised digesta v. frozen digesta, with no difference in S. bovis levels. These data indicate that for these bacteria at least, faeces are a suitable model for studying the bacterial ecosystem within the equine colon. The present study also indicates that the preservation method of digesta affects levels of bacteria detected.

Expression of mRNA for galanin, galanin like peptide and galanin receptors 1-3 in the ovine hypothalamus and pituitary gland: effects of age and gender.

The neurotransmitters/neuromodulators galanin (GAL) and galanin-like peptide (GALP) are known to operate through three G protein-coupled receptors, GALR1, GALR2 and GALR3. The aim of this study was to investigate changes in expression of mRNA for galanin, GALP and GALR1-3 in the hypothalamus and pituitary gland, of male and female sheep, to determine how expression changed in association with growth and the attainment of reproductive competence. Tissue samples from the hypothalami and pituitary glands were analysed from late foetal and pre-pubertal lambs and adult sheep. Although mRNA for galanin and GALR1-3 was present in both tissues, at all ages and in both genders, quantification of GALP mRNA was not possible due to its low levels of expression. mRNA expression for both galanin and its receptors was seen to change significantly in both tissues as a function of age. Specifically, hypothalamic galanin mRNA expression increased with age in the male, but decreased with age in the female pituitary gland. mRNA expression for all receptors increased between foetal and pre-pubertal age groups and decreased significantly between pre-pubertal and adult animals. The results indicate that the expression of mRNA for galanin and its receptors changes dynamically with age and those significant differences exist with regard to tissue type and gender. These changes suggest that galaninergic neuroendocrine systems could be involved in the regulation of ovine growth and or the development of reproductive competence. The roles played by these systems in the sheep, however, may differ from other species, in particular the neuroendocrine link between nutrition and reproduction and GALR1s role in pituitary signalling.

Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factor binding proteins in sheep

Evidence suggests that the insulin-like growth factor binding proteins (IGFBPs) are involved in modulating the role that IGF-I and -II play in regulating follicular growth and development in sheep. However, little information exists as to the role that key peripheral factors play in regulating the expression of IGFBP components within the follicle. The present study investigated the regulatory effects of FSH and LH on gene expression for IGFBP-2 to -6 in ovine follicles, using bovine follicular fluid (bFF) and gonadotrophin-releasing hormone antagonist (GnRHa) model systems to perturb endogenous gonadotrophin secretion. Gene expression studies were carried out using in situ hybridisation with sheep-specific ribonucleotide probes. Treatment of ewes with bFF had few consistent effects on IGFBP-3 and -4 mRNA expression in follicles, whereas bFF treatment resulted in significant decreases in IGFBP-2 and IGFBP-6 mRNA expression in only medium follicles 60 h after onset of bFF, and in follicles 12h after the onset of bFF, respectively. Treatment of ewes with GnRHa resulted in transient significantly increased IGFBP-2 mRNA in healthy follicles in comparison to early atretic or atretic follicles 12h post-GnRHa (P<0.05), in IGFBP-3 mRNA in early atretic and atretic follicles 36 h post-GnRHa (P<0.001), and IGFBP-5 mRNA in follicles 12h post-GnRHa (P<0.001). In contrast, GnRHa treatment resulted in significant decreases in IGFBP-4 (P<0.001) and IGFBP-6 (P<0.01) mRNA expression in large follicles 36 h post-GnRHa, and atretic follicles by 60 h post-GnRHa, respectively. These data highlight that FSH and LH are involved, at least in part, in mediating the proliferative and differentiative changes in intrafollicular IGFBP levels that are observed during follicular growth and atresia in the sheep.

Effects of cycle stage on regionalised galanin, galanin receptors 1–3, GNRH and GNRH receptor mRNA expression in the ovine hypothalamus

The neurotransmitter galanin has been implicated in the steroidogenic regulation of reproduction based on work mainly conducted in rodents. This study investigated the temporal changes in the expression of galanin and its three receptor isoforms and GNRH and GNRHR mRNA in specific hypothalamic nuclei known to be involved in the regulation of reproductive cyclicity, namely the medial pre-optic area (mPOA), the rostral mPOA/organum vasculosum of the lamina terminalis, the paraventricular nucleus and the arcuate nucleus using an ovine model. Following synchronisation of their oestrous cycles, tissues were collected from ewes at five time points: the early follicular, mid follicular (MF) and late follicular phases and the early luteal and mid luteal phases. The results indicated significant differences in regional expression of most of the genes studied, with galanin mRNA expression being highest during the MF phase at the start of the GNRH/LH surge and the expression of the three galanin receptor (GalR) isoforms and GNRH and its receptor highest during the luteal phase. These findings are consistent with a role for galanin in the positive feedback effects of oestradiol (E(2)) on GNRH secretion and a role for progesterone induced changes in the pattern of expression of GalRs in the regulation of the timing of E(2)s positive feedback through increased sensitivity of galanin-sensitive systems to secreted galanin.

Effects of probiotic Enterococcus faecium and Saccharomyces cerevisiae on the faecal microflora of pet rabbits

OBJECTIVES Probiotics are frequently used in the treatment of gastrointestinal diseases in pet rabbits based largely on anecdotal evidence of a beneficial effect. However, there has been little work performed to assess any such benefit in health or disease. The aim of this study was to determine the effect of probiotics on faecal levels of four important candidate gastrointestinal bacteria (Bacteroides species, Enterococcus faecium, Fibrobacter succinogenes and Clostridium spiroforme) in pet rabbits. Additional aims were to evaluate the effect of probiotics on bodyweight and faecal weight and diameter. MATERIALS AND METHODS Double-blind triple cross-over study in six healthy rabbits orally administered two probiotic strains, Saccharomyces cerevisiae NCYC Sc47 and E. faecium NCIMB 30183. Levels of bacteria in faecal pellets were subsequently determined by real-time quantitative polymerase chain reaction. RESULTS Oral administration of probiotic E. faecium NCIMB 30183 was associated with a significant (P = 0 · 042) increase in faecal levels of E. faecium. However, probiotic treatment did not affect faecal levels of Bacteroides species, F. succinogenes or C. spiroforme, bodyweight, or faecal weight and diameter. CLINICAL SIGNIFICANCE The inclusion of dietary probiotic supplementation using E. faecium NCIMB 30183 can increase faecal levels of certain bacterial flora of healthy adult rabbits. Further work is required to investigate the effects of probiotics in animals affected with gastrointestinal disease.

Developmental programming: prenatal androgen exposure alters the gonadotroph population of the ovine pituitary gland.

In utero exposure of the female foetus to androgens during development disrupts the reproductive axis and results in hypersecretion of luteinising hormone (LH) (but not follicle‐stimulating hormone) in postnatal life. Abnormalities in the neural circuits controlling hypothalamic gonadotrophin‐releasing hormone have been documented; however, androgens could also programme abnormalities in the pituitary gland. Ovine foetuses were exposed to either testosterone propionate or the non‐aromatisable androgen dihydro‐testosterone from days 30–90 of gestation (term 147 days) and the effects on the functional morphology of the pituitary were determined. Exogenous testosterone propionate exposure resulted in pituitary glands in adult male and female sheep that were 40% heavier than controls. Because this effect was not observed in the dihydro‐testosterone‐exposed animals, these actions are mediated via the oestrogen receptor (ER). No significant differences were apparent in 90‐ or 140‐day foetuses. There was no difference between control and androgen‐exposed animals in the density of LHβ or ERα immunoreactive cells in the pituitary although the density of follicle‐stimulating hormone‐β immunoreactive cells was lower in the testosterone‐treated animals. The percentage of cells co‐localising LHβ and ERα was lower in the testosterone‐treated ewes and this may, in part, explain a reduced ability to respond to steroid feedback. Thus, enlargement of the pituitary gland, coupled with a reduced sensitivity to oestrogen negative‐feedback, may contribute to the hyper‐secretion of LH observed in animals that have been exposed to excess androgens during foetal life.

Early post parturient changes in milk acute phase proteins.

The periparturient period is one of the most critical periods in the productive life of a dairy cow, and is the period when dairy cows are most susceptible to developing new intramammary infections (IMI) leading to mastitis. Acute phase proteins (APP) such as haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) have been detected in milk during mastitis but their presence in colostrum and milk in the immediate postpartum period has had limited investigation. The hypothesis was tested that APP are a constituent of colostrum and milk during this period. Enzyme linked immunosorbent assays (ELISAs) were used to determine each APPs concentration in colostrum and milk collected daily from the first to tenth day following calving in 22 Holstein-Friesian dairy cows. Haptoglobin was assessed in individual quarters and composite milk samples while M-SAA3 and CRP concentration were determined in composite milk samples. Change in Hp in relation to the high abundance proteins during the transition from colostrum to milk were evaluated by 1 and 2 dimension electrophoresis and western blot. In 80% of the cows all APPs were detected in colostrum on the first day following parturition at moderately high levels but gradually decreased to minimal values in the milk by the 6th day after calving. The remaining cows (20%) showed different patterns in the daily milk APP concentrations and when an elevated level is detected could reflect the presence of IMI. Demonstration that APP are present in colostrum and milk following parturition but fall to low levels within 4 days means that elevated APP after this time could be biomarkers of post parturient mastitis allowing early intervention to reduce disease on dairy farms.

Masculinization of the Distal Tubular and External Genitalia in Female Sheep With Prenatal Androgen Exposure

Prenatal exposure to endogenous or exogenous androgens alters the development of the female reproductive tract. Although lesions in ovaries and external genitalia of androgenized female sheep have been reported, lesions of the tubular genitalia have not. Testosterone propionate (TP) or dihydrotestosterone (DHT) was administered by intramuscular injection twice weekly to 32 ewes from 30 to 90 days of pregnancy. The ewes lambed normally. The reproductive tracts from 24 treated and 13 control postpubertal female offspring were examined at 10 months of age. The ovaries, oviducts, and uteri were grossly and histologically normal in both TP- and DHT-exposed sheep. However, in the DHT-treated sheep, the uterus connected to a misshapen, saccular vagina that opened into the urethra; in the TP-treated sheep, it ended in a blind sac. In both TP- and DHT-treated sheep, the urethra was approximately 5 times longer than that of control sheep, and it resembled a male urethra with bilateral male accessory genital glands. The urethra terminated in a fully developed penis in both TP- and DHT-treated sheep, and a scrotal sac was present (without testes). These results show that prenatal exposure of female sheep to exogenous androgens results in masculinization of the tubular and external genitalia.

Equine dietary supplements: an insight into their use and perceptions in the Irish equine industry

BackgroundNutritional supplements are frequently used by horse owners/caregivers to supplement their horse(s) diets. Some work has been done to identify the types of supplements fed and the reasons for doing so; however, this has been predominantly disciple-specific and with little focus on participants’ perceptions of supplement testing and regulation. The aim of this study was to gain an insight into the use and perceptions of equine dietary supplements in the Irish equestrian industry.MethodsAn online survey was designed to ascertain the following information: demographics, types of supplements fed and reasons for use, factors that influenced respondents’ choice of supplement, where advice was sought and perceptions of testing and regulation of equine supplementsResultsThe survey yielded 134 responses, 70% non-professionals and 30% professionals. A greater percentage of professionals included supplements in their horse(s) diets (98%) compared to non-professionals (86%). Almost 70% of professionals fed more than two supplements, whereas 80% of non-professionals reported to feed only one supplement. Joint supplements were most commonly fed by all respondents (22%) followed by calming supplements (13%). The enhancement of performance (35%) and prevention of joint disorders (34%) were the most common reasons reported by respondents for using a supplement. Over 53% of respondents sought advice on choosing a supplement from their feed merchant, followed by their veterinarian (46%). Veterinary recommendation was given as the most influential factor when choosing a supplement by 90% of respondents, followed by cost (69%). Most (93%) respondents thought that feed supplements had to meet legal standards, with each batch analysed for quality (72%) and the supplement tested on horses before being launched on to the market (92%).ConclusionThis study has identified the main types of supplements used in the Irish equestrian industry along with the reasons for their use. However, it has also highlighted major misperceptions in how supplements are tested before being launched for sale and further work on this aspect of the findings would be beneficial.