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Topic:Biochemistry
The study of biochemistry in horses encompasses the chemical processes and substances that occur within equine organisms. This field investigates the molecular interactions and pathways that are fundamental to horse physiology, including metabolism, enzyme activity, and genetic expression. Key areas of interest include the examination of metabolic disorders, nutrient absorption, and the biochemical basis of muscle function and energy production. Researchers utilize biochemical analysis to understand health and disease mechanisms in horses, contributing to the development of diagnostic tools and therapeutic strategies. This page gathers peer-reviewed studies and scholarly articles that explore various biochemical processes and their implications for equine health and performance.
A new method for quantitation of spin concentration by EPR spectroscopy: application to methemoglobin and metmyoglobin. A new method of EPR spectral analysis is developed to quantitate overlapping signals. The method requires double integration of a number of spectra containing the signals in different proportions and the subsequent solution of a system of linear equations. The result gives the double integral values of the individual lines, which can then be further used to find the concentrations of all the paramagnetic species present. There is no requirement to deconvolute the whole spectrum into its individual components. The method is employed to quantify different heme species in methemoglobin and metmyo...
Expression of CRISP proteins in the male equine genital tract. Cysteine rich secretory proteins (CRISPs) have been detected immunochemically in the equine male genital tract. CRISPs are secretory products of the epididymis, the ampulla and the seminal vesicle. A particular feature of the horse is the abundance of CRISPs in seminal plasma. CRISPs can also be detected in extracts of testicular, epididymal and ejaculated spermatozoa in increasing amounts. Unlike other seminal plasma proteins, they cannot be removed completely from spermatozoa by high salt treatment. The remaining CRISP antigens are localized on the midpiece, and the postacrosomal and equator...
Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80-90% methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system,...
Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation. Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the m...
Muscle adaptation of Thoroughbred racehorses trained on a flat or sloped track. To determine histochemical and biochemical properties of muscle during adaptation to training on a flat or sloped track. Methods: 22 Thoroughbreds. Methods: Samples were obtained from the middle gluteus muscle before and after training programs were conducted, using a needle-biopsy technique. Training programs consisted of horses running 1,600 m on a flat or sloped track for 16 weeks. Amplitude of middle gluteus muscle activity per burst was calculated. Muscle fiber composition and area were examined on serial cross sections processed by standard histochemical staining procedures (ATPase stain...
Determination of testosterone:epitestosterone ratio after pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation using gas chromatography-mass spectrometry in equine urine. A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of ...
Gastrointestinal and endocrine function during ‘foal heat diarrhoea’ in healthy foals. Gastrointestinal function was assessed in normal foals before, during and after the onset of 'foal heat diarrhoea' by haematological, biochemical, microbiological and parasitological methods, and by a combined lactose tolerance-breath hydrogen excretion test. Warmblood mares (n=11) and their foals were used. The foals were assessed according to a standard protocol after birth and at regular intervals thereafter. All except one of the foals suffered from diarrhoea that started between day 8 and day 12 after birth, which lasted for 1-4 days. Rotavirus was detected in the faeces of five foals, bu...
The effects of oxytocin and progestagens on myometrial contractility in vitro during equine pregnancy. Progesterone or progestagens are thought to maintain myometrial quiescence in pregnant mares, although this has not been proven. In the present study, the contractility of the equine myometrium was tested in vitro using samples collected from pregnant mares (n=33) between day 68 and day 340 of gestation. Myometrial samples were equilibrated in aerated Krebs buffer and subjected to one or more of these treatments: (i) oxytocin only; (ii) initial oxytocin treatment followed by combined oxytocin and progesterone or another progestagen; and (iii) initial oxytocin treatment followed by Krebs buffer...
Changes in PGF2alpha secretion during prolonged luteal phase in mares. The aim of this study was to characterize changes in PGF2alpha secretion in mares with persistent corpora lutea that were induced by administering altrenogest during oestrus. In Expt 1, PGF2alpha secretion was compared among mares undergoing normal oestrous cycles (n=7) and mares undergoing prolonged luteal phases (n=6), using the mean 15-ketodihydro-PGF2alpha (PGFM) plasma concentrations, peak PGFM concentrations and number of PGFM surges each day, from day 12 to day 16 of the luteal phase. In Expt 2, oxytocin-induced PGF2alpha secretion was characterized on days 13 and 16 of the luteal phase...
Identification of proteins in the equine embryonic capsule. An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like g...
Ovarian secretion of inhibin in mares. In mares, circulating immunoreactive inhibin concentrations increase during the follicular phase and decrease at the start of the LH surge. Thereafter, sharp increases in circulating immunoreactive inhibin concentrations, the 'ovulatory increase', are observed during ovulation. In the present study, the cellular sources and molecular form of ovarian inhibin were investigated to determine the mechanism responsible for this unique ovulatory increase. Three sizes of ovarian follicles (small, 30 mm in diameter) were selected. Inhibin alpha-subunit was localized by immunohistochemistry to the gran...
Differential gene expression in day 12 and day 15 equine conceptuses. Complex changes in gene expression must occur at the proper time and in the appropriate tissues for pregnancy to be successful. Therefore, research aimed at defining the regulation of gene expression in conceptuses is of critical importance. However, information on developmentally regulated changes in gene expression in horse conceptuses is sparse and inadequate. In the present study, suppression subtractive hybridization was used to identify genes that are expressed more highly at day 15 than on day 12 of gestation. This period encompasses maternal recognition of pregnancy and the beginning o...
Differential expression of steroidogenic enzymes by the primary corpora lutea of pregnant mares during equine chorionic gonadotrophin secretion. At the onset of equine chorionic gonadotrophin (eCG) secretion, eCG stimulates luteal androgen and oestrogen production. Although eCG concentrations increase exponentially from day 37 to day 60 of gestation and eCG is detectable in maternal serum until about day 120-150 of gestation, luteal androgen and oestrogen production peaks between 5 and 10 days after initial exposure to eCG and then decreases gradually. It is not clear how eCG regulates luteal androgen and oestrogen production. In the present study, the steady-state mRNA expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cyto...
Effect of cryopreservation and oviductal cell conditioned media on Ca2+ flux of equine spermatozoa. Movement of Ca2+ into spermatozoa is a critically important event for capacitation and the acrosome reaction. In the present study, the nature of Ca2+ movement in fresh equine spermatozoa was established and the effects of oviductal cell conditioned medium (OCM) and cryopreservation on Ca2+ flux were investigated. The ability of fresh and cryopreserved stallion spermatozoa to regulate Ca2+ concentration over time was evaluated in Ca2+ -free PBS. Intracellular Ca2+ concentrations were higher in cryopreserved spermatozoa than in fresh spermatozoa. However, extracellular Ca2+ concentrations were ...
Biophysical and 1H magnetic resonance spectroscopy characteristics of fractionated stallion ejaculates. The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was use...
Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions. In the present study, the interactions among stallions, semen extenders and cooling treatments before stallion semen samples were frozen were studied. In Expt 1, the effects of four cooling treatments and three semen extenders were investigated (11 stallions x four split ejaculates), whereas in Expt 2, the effects of two semen extenders, two egg yolk concentrations and two glycerol concentrations were investigated (six stallions x five split ejaculates). Sperm motility after thawing was evaluated. In Expt 1, the extender x cooling treatment interaction was significant. Centrifugation and addit...
Testicular secretion of inhibin in stallions. In this study, clear seasonal changes in the circulating concentrations of immunoreactive inhibin were observed in stallions throughout the year. The highest plasma concentrations of immunoreactive inhibin were observed during the breeding season and the lowest concentrations were observed during the nonbreeding season. There was positive correlation between the circulating concentrations of immunoreactive inhibin, steroid hormones and gonadotrophins. Inhibin alpha subunit positive staining was observed in Sertoli cells and more clearly in Leydig cells. Both cell types also stained positively ...
Influence of the dopamine antagonist domperidone on the vernal transition in seasonally anoestrous mares. The aim of the present study was to determine the effect of prolonged administration of the dopamine antagonist domperidone on follicular development, ovulation and endocrine profiles in anoestrous mares. Anoestrous mares (n=16) were maintained under natural photoperiod and ambient temperature. Eight of the mares were treated with domperidone each day from 15 January until the first ovulation of the year. The mean number and size of follicles > or =20 mm in diameter were significantly greater in domperidone-treated mares than in control mares by day 14 of treatment. The day of first ovulati...
Immunolocalization of oxytocin and neurophysin in the mare uterus. The presence of oxytocin in the mare uterus was determined by radioimmunoassay of tissue extracts and uterine lavage fluid, and by immunohistochemistry. Samples were obtained from mares during the oestrous cycle and on day 14 of pregnancy. Immunoreactive oxytocin was detected in extracts of endometrium and myometrium, and in uterine lavage fluid. In tissue sections, the luminal epithelium, the epithelium of the superficial, but not deep, glands, blood vessel walls and myometrium all stained positively for oxytocin, whereas only the luminal epithelium and epithelium of the superficial glands st...
Relationship between sperm nuclear protamine free -SH status and susceptibility to DNA denaturation. Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples...
Effect of oestradiol on LH secretion and pituitary responsiveness to GnRH in ovariectomized mares. Long-term ovariectomized Pony mares were treated with oestradiol (0.2-5.0 mg; i.m.) at 12 h intervals for 10 days. Blood samples were collected by jugular venepuncture three times a day throughout the experiment and additional blood samples were collected at 15 min intervals for 12 h on days 0 and 10 (sampling periods 1 and 2, respectively). There were significant effects of oestradiol treatment (P < 0.05) and oestradiol treatment x day (P < 0.0001) on the mean LH concentrations each day. Regression analysis of LH time trends each day indicates that there is heterogeneity (P < 0.001) ...
Immunolocalization of a novel protein (P19) in the endometrium of fertile and subfertile mares. One of the major progesterone-dependent endometrial proteins in the mare is a novel 19 kDa lipocalin (P19). This protein is secreted by the endometrial glands and is readily detectable in uterine secretions during the luteal phase of the oestrous cycle and early pregnancy. The function of P19 is unknown, but since most lipocalins act as carriers of small hydrophobic molecules, it probably transports a maternal factor to the conceptus during pregnancy. In this study, a high titre antiserum raised against recombinant-derived P19 was used to detect by immunohistochemistry the protein in endometri...
Localization and cellular distribution of a unique hyaluronidase in stallion spermatozoa during epididymidal transit. Three protein bands with hyaluronidase activity and molecular masses of 87, 48 and 43 kDa were isolated from purified equine sperm plasma membranes. Indirect immunofluorescence was used to assess sperm labelling patterns using a polyclonal antibody to sperm hyaluronidase. In ejaculated spermatozoa, surface-associated hyaluronidase was localized to the posterior head region of 98 +/- 2% of spermatozoa (n=10). Epididymides were isolated from mature stallions (n=5) and divided into caput, corpus and cauda epididymides in separate Petri dishes. The epididymidal tubules were dissected and washed us...
The role of endogenous opioids in the ovulatory LH surge in mares. Removal of opioid inhibition of GnRH neurones is thought to be a critical event in generating the ovulatory surge in some species. In the present study, a nonsurgical technique was used to collect pituitary venous blood samples from eight mares every 0.5-1.0 min for 1 h before and after administration of the opioid receptor antagonist naloxone (0.2 mg kg(-1), i.v.), to investigate whether opioid inhibition is also important in mares. Jugular blood samples were taken at 10-15 min intervals. Mares were studied 0, 1 or 2 days before ovulation. Naloxone administration increased mean rates of GnRH ...
Partial characterization of an equine conceptus prostaglandin inhibitory factor. Equine conceptuses are thought to produce antiluteolytic factors that inhibit endometrial PGF2alpha and, thus, prevent luteolysis in pregnant mares. The aim of the present study was to characterize partially the chemical nature of the prostaglandin inhibitory factor (PIF) produced by equine conceptuses in vitro. Embryos were collected from pregnant mares 13 +/- 0.5 days after ovulation and were cultured for 24 h. Harvested equine conceptus conditioned media (CCM) were assayed for antiluteolytic activity by determining the inhibition of endometrial PGF2alpha synthesis in vitro. Significant anti...
Apoptosis in equine granulosa cells and its relationship to cumulus expansion and oocyte chromatin configuration in ovarian follicles. During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa ...
Indirect determination of stallion sperm capacitation based on esterase release from spermatozoa challenged with lysophosphatidylcholine. A spectrophotometric assay was developed to measure the amount of esterase released from stallion spermatozoa. This assay was used to determine the percentages of capacitated stallion spermatozoa, determined by the ability of spermatozoa to undergo an acrosome reaction and release esterase in response to a lysophosphatidylcholine challenge, for spermatozoa incubated under conditions to increase intracellular calcium and cAMP. Incubation with 100 nmol calcium ionophore A23187 l(-1) induced 66% of stallion spermatozoa to capacitate after 60 min of incubation at 37 degrees C. Subsequent experimen...
Estimation of sodium and potassium concentrations in the uterine fluid of mares by microdialysis and ion chromatography. Equine blastocyst fluid has a lower [Na+], a higher [K+] and a lower osmolality than does normal blood serum. Based on the assumptions that the sodium pump is primarily responsible for fluid accumulation and that ions transported actively into the blastocyst increase blastocyst osmolality above that of the external medium, we hypothesized that the [Na+] and the osmolality of mare uterine fluid are lower than those of blastocyst fluid. Microdialysis and ion chromatography were used to estimate [Na+] and [K+] of uterine fluid. Mares (n=10) were used for in vivo measurements at different stages o...
The uterus as a source of oxytocin in cyclic mares. In a recent study, continuous administration of oxytocin by subcutaneous minipump to mares from day 8 to 20 after ovulation prevented luteolysis in most of the treated but none of the control mares, indicating a role for oxytocin in cyclical luteolysis in mares. In the present study, measurement of oxytocin concentrations in uterine flushings recovered from nine mares during days 14-18 after ovulation gave values that were many times higher than those measured concurrently in peripheral plasma. Furthermore, intrauterine administration of oxytocin to four mares on day 14 after ovulation stimula...
Preservation of stallion sperm quality by native phosphocaseinate: a direct or indirect effect? Milk-based diluents are generally considered efficient for survival of stallion spermatozoa in vitro. However, milk is a complex and variable medium and native phosphocaseinate is a milk component that is more efficient for preservation of sperm motility and fertility, although the mechanisms involved in this protection have not yet been elucidated. The aim of the present study was to characterize the interactions between native phosphocaseinate and equine spermatozoa. No binding between sperm membranes and native phosphocaseinate was observed using indirect immunofluorescent staining or elect...
