Sulfamethoxazole biodegradation and impacts on soil microbial communities in a Bolivian arid high altitude catchment.

The processes controlling antibiotics fate in ecosystems are poorly understood, yet their presence can inhibit bacterial growth and induce the development of bacterial resistance. Sulfamethoxazole (SMX) is one of the most frequently detected sulfonamides in natural environments due to its low metabolism and molecular properties. This work presents pioneering results on SMX biodegradation and impact in high altitude soils (Bolivian Altiplano), allowing a better understanding of the persistence, spread and impact of this antibiotic at the global watershed scale. Our results showed significant dissipation of SMX in relation to its adsorption, hydrolysis and biotransformation. However, biodegradation appears to be lower in these mountain soils than in lowland soils as widely described in the literature. The half-life of SMX ranges from 12 to 346 days in non-sterile soils. In one soil, no biotic degradation was observed, indicating a likely high persistence. Biodegradation was related to OC content and to proximity to urban activities. Regarding the study of the impacts of SMX, the DGGE results were less sensitive than the sequencing. In general, SMX strongly changes the structure and composition of the studied soils at high altitudes, which is comparable to the observations of other authors in lowland soils. The phylum Actinobacter showed high sensitivity to SMX. In contrast, the abundance of ɣ-proteobacteria remained almost unchanged. Soil contamination with SMX did not lead to the development of the studied resistance genes (sul1 and sul2) in soils where they were absent at the beginning of the experiment. Thus, the presence of SMX resistance genes seems to be related to irrigation with wastewater carrying the studied resistance genes.

Assessment of the Sulfamethoxazole mobility in natural soils and of the risk of contamination of water resources at the catchment scale.

Sulfamethoxazole (SMX) is one of the antibiotics most commonly detected in aquatic and terrestrial environments and is still widely used, especially in low income countries. SMX is assumed to be highly mobile in soils due to its intrinsic molecular properties. Ten soils with contrasting properties and representative of the catchment soil types and land uses were collected throughout the watershed, which undergoes very rapid urban development. SMX displacement experiments were carried out in repacked columns of the 10 soils to explore SMX reactive transfer (mobility and reactivity) in order to assess the contamination risk of water resources in the context of the Bolivian Altiplano. Relevant sorption processes were identified by modelling (HYDRUS-1D) considering different sorption concepts. SMX mobility was best simulated when considering irreversible sorption as well as instantaneous and rate-limited reversible sorption, depending on the soil type. SMX mobility appeared lower in soils located upstream of the watershed (organic and acidic soils - Regosol) in relation with a higher adsorption capacity compared to the soils located downstream (lower organic carbon content - Cambisol). By combining soil column experiments and soil profiles description, this study suggests that SMX can be classified as a moderately to highly mobile compound in the studied watershed, depending principally on soil properties such as pH and OC. Potential risks of surface and groundwater pollution by SMX were thus identified in the lower part of the studied catchment, threatening Lake Titicaca water quality.

Antibiotic pollution in the Katari subcatchment of the Titicaca Lake: Major transformation products and occurrence of resistance genes.

An increasing number of studies pointed out the ubiquitous presence of medical residues in surface and ground water as well as in soil compartments. Not only antibiotics can be found in the environment but also their transformation products about which little information is generally available. The development of bacterial resistance to antibiotics is particularly worrying as it can lead to sanitary and health problems. Studies about the dissemination of antibiotics and associated resistances in the Bolivian Altiplano are scarce. We provide baseline information on the occurrence of Sulfamethoxazole (SMX) and Trimethoprim (TMP) antibiotics as well as on the most common human SMX transformation products (TP) and on the occurrence of sulfonamide resistance genes. The studied water and soil compartments presented high levels of antibiotic pollution. This situation was shown to be mainly linked with uncontrolled discharges of treated and untreated wastewaters, resulting on the presence of antibiotics in the Titicaca Lake. SMX TPs were detected in surface waters and on soil sampled next to the wastewater treatment plant (WWTP). SMX resistance genes sulI and sulII were widely detected in the basin hydrological network, even in areas unpolluted with antibiotics. Mechanisms of co-selection of antibiotic- and metal- resistance may be involved in the prevalence of ARG's in pristine areas with no anthropogenic activity and free of antibiotic pollution.

Asynchronous ovulation in mares: seasonal variations in frequency.

The mare's reproductive system is rarely capable of sustaining multiple pregnancies to term. Multiple pregnancies largely derive from multiple ovulations, most commonly double ovulations, hence, double ovulations are of significant concern to breeders/veterinarians. Double ovulations may be synchronous or asynchronous. Asynchrony of up to 96 hours may result in two embryonic vesicles of very different sizes, which are challenging to detect at early ultrasonic pregnancy detection. This study aims to (1) document the incidence of synchronous and asynchronous double ovulation and (2) determine whether this varies with month/season. 506 cycles from double ovulating mares were monitored at 8±1 hour intervals before expected ovulation until 96 hours post initial ovulation. Mares were grouped according to eight-hour ovulation intervals and month/season. When asynchrony was classed as greater than or equal to eight hours or greater than or equal to 24 hours apart, respectively, a significant (P<0.001) difference existed between the number of mares demonstrating asynchronous double ovulations (65.8 and 28.5 per cent) and synchronous double ovulations (34.2 and 71.5 per cent). Significantly (P<0.05), more asynchronous ovulations occurred in the seasonal transition periods. This study demonstrates that asynchronous double ovulation is common and emphasises the importance of closely monitoring mares, particularly at the extremes of the breeding season, for double ovulation up to or at 96 hours postinitial ovulation in order to minimise the chances of missing multiple pregnancy.

Effect of heterologous and homologous seminal plasma on stallion sperm quality.

Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to investigate the effect of adding back SP to colloid centrifuged spermatozoa to determine its effect on spermatozoa; and (2) to investigate whether the stallion's own SP had a greater or lesser effect than heterologous SP. Conventional semen doses were sent from a stud overnight to the laboratory using standard transport conditions. Once at the laboratory, the semen samples were used for single layer centrifugation with Androcoll-E, and the resulting sperm preparations were treated with heterologous SP. Adding SP had a small but significant effect on sperm motility but no effect on the proportion of spermatozoa that had acrosome reacted. There were significant increases in hydrogen peroxide production and chromatin damage (P < 0.001). When homologous and heterologous SP were compared, considerable variation was observed between stallions, so that it was not possible to predict whether homologous or heterologous SP, or no SP, will produce the best motility for spermatozoa from any given stallion. Therefore, it is necessary to test different combinations of spermatozoa and SP to find the optimal effect on motility. The SP from most stallions increased reactive oxygen species and chromatin damage. In conclusion, the interaction between SP and spermatozoa depends on the origin of both SP and spermatozoa. If it is desirable to add SP to stallion sperm samples, it should be done directly before insemination rather than before storage, because of increased hydrogen peroxide production and sperm chromatin damage.

Equine endometrial cytology and bacteriology: effectiveness for predicting live foaling rates.

Endometritis is an important cause of sub-fertility in mares. The critical indicator of reproductive success and financial return for commercial studs is live foaling rate. Endometrial bacteriology and/or cytology are used to diagnose endometritis and thus identify mares at risk of early embryonic death. However, mares with endometritis may conceive but then abort in late gestation. The aims of this study were to establish, as part of a standard breeding examination (1) whether a threshold percentage of uterine polymorphonuclear neutrophils (PMNs) exists above which a significant reduction in live foaling rate is evident; (2) the relationship of a positive bacteriology result to live foaling rate, and (3) the relationship of a combination of positive cytology and bacteriology result to live foaling rate. Guarded endometrial swabs (n=2660) were collected from 1621 Thoroughbred mares on 17 commercial stud farms by five veterinarians during a single breeding season. All mares were included regardless of age, history or parity. Cytological and bacteriological analyses were performed on each swab and subsequent live foaling rates recoded. Data were analysed by comparing 0%, ≥ 1%, ≥ 2%, ≥ 5% or ≥ 25% PMNs of total cells counted, or categories of bacterial growth to live foaling rates, using Pearson's chi-squared test. A threshold value of ≥ 1% PMNs, culture of a single bacterial isolate and a combination of both these parameters were associated with significantly reduced live foaling rates. Positive cytology alone, positive bacterial culture alone, or combined positive cytology and bacteriology were equally indicative of the likelihood of a mare producing a live foal.

Manual reduction of multiple embryos in the mare: the effect on subsequent pregnancy outcome.

To determine whether manually reduced multiple pregnancies (MPs) are at a greater risk of pregnancy loss than single pregnancy (SP) in mares, and to examine if a difference exists in the timing of pregnancy loss between manually reduced MPs and SPs, 1916 Thoroughbred mares were ultrasonically monitored every 2 days during oestrus to confirm ovulation, and up to Day 40 post-ovulation to confirm pregnancy. Ultimate pregnancy outcome was ascertained from the General Thoroughbred Stud book and classified as live foal (LF), early abortion (EA; Days 40-150), slipped foal (SF; Days 150-term), and barren (B; Time of pregnancy loss unknown). Significantly (P<0.05) more SPs failed (17.23%; 226/1312) than manually reduced MPs (13.41%; 81/604). Both SPs and MPs were at greatest risk of being lost as EA (72.16%; 127/176 and 61.67%; 37/60, respectively) compared to SF (27.84%; 49/176 and 38.33%; 23/60). There was no significant difference in the time of greatest risk of pregnancy loss between manually reduced MPs and SPs. It was concluded that owners and veterinarians can be assured that manual reduction of MPs does not increase the risk of pregnancy loss above those associated with SPs.

Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation).

The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Thoroughbred mares, aged 2-26 years, were examined with ultrasound to ascertain ovulation date to within 24h, and pre-ovulatory follicle(s) (F1) diameter. Mares were divided into groups according to age (7 groups, 2-4 yr, 5-7 yr, 8-10 yr, 11-13 yr, 14-16 yr, 17-19 yr, >19 yr), time within the season (16 half-month groups, from Feb 1(st) to Sept 30(th)), and pre-ovulatory follicles (single, {SO} or multiple {MO}). Overall average F1 diameter was 39.95 ± 4.84 mm (range 22-50 mm). Mare age had a significant (P < 0.001) negative effect on F1 diameter (largest F1 38.95 ± 5.61 mm, mares 2-4 yrs; smallest F1 33.30 ± 4.66 mm, mares >19 yrs) as did season (largest F1 44.20 ± 3.95 mm, Feb 1(st)-14(th); smallest F1 33.74 ± 4.87 mm, Aug 15(th)-31(st)) and the presence of more than one pre-ovulatory follicle (MO F1 35.45 ± 4.53 mm; SO F1 37.44 ± 4.84 mm). In conclusion older mares, bred towards the end of the breeding season, especially if MO were present, were more likely to ovulate from smaller follicles. If, as suggested, small pre-ovulatory follicle size is associated with low oocyte viability, then this may account, at least in part, for the poor fertility rates characteristic of older MO mares, bred later in the season and so justify increased monitoring and careful reproductive management of such mares.

Factors affecting the incidence of postpartum oestrus, ovarian activity and reproductive performance in Thoroughbred mares bred at foal heat under Indian subtropical conditions.

Decreased reproductive performance due to summer stress is a well known phenomenon in farm livestock. Whether this occurs in the mare and specifically how this might affect postpartum reproductive activity and performance, especially at Foal Heat (FH), is unknown. This study, therefore, aims to investigate this and the factors that might affect postpartum reproductive activity. Reproductive records of 228 Thoroughbred mares (694 mare years) bred in subtropical north-western India were retrospectively analysed. Overt oestrous activity occurred within 21 d postpartum in 92.94% (645/694) of mares. Significantly (p<0.001) more April foaling mares (97.37%, 185/190) expressed postpartum oestrous activity than those foaling in January (83.61%; 51/61) and February (88.49; 123/139). Similarly significantly (p<0.01) fewer multiparous mares failed to demonstrate oestrous activity than primiparous mares (6.12% vs.15.07%; 38/621 vs. 11/73, respectively). 190 of these 694 mares were additionally monitored to confirm ovulation; in these mares onset of FH (oestrus plus confirmed ovulation) occurred 8.42+/-0.17 d and first ovulation 13.64+/-0.20 d postpartum. Month, stud farm, year, and parity did not affect interval from parturition to FH onset or to first ovulation; or FH onset to ovulation. In FH bred mares Day 16 pregnancy rate and overall foaling rate were 53.76% (100/186) and 46.24% (86/186) respectively and were similar to those of mares bred later postpartum. FH pregnancy rates were not affected by stud, season, month, year, number of matings, or day of ovulation but were significantly (p<0.008) lowered by increasing mare age. Significantly (p<0.01) lower Day 16 pregnancy rates were observed in uterine treated mares compared to untreated mares (31.09% vs. 57.96%; 9/29 vs. 91/157, respectively), this difference was not evident during the rest of pregnancy. In conclusion, postpartum reproductive and ovarian activity appears to be affected by environment, i.e., delayed in subtropical kept Thoroughbred mares compared to those kept in temperate climates. However, resulting reproductive performance at FH and the factors affecting postpartum reproductive activity are similar.

The pattern of embryonic fixation and its relationship to pregnancy loss in thoroughbred mares.

Ultrasonographic pregnancy records of 195 mares from six Thoroughbred stud farms, over a period of 7 years were retrospectively analysed to assess the effect of various factors on embryonic vesicle (EV) fixation pattern and pregnancy outcome. Of the total of 746 pregnancies analysed, significantly (p < 0.01) more EV fixations were evident in the right uterine horn than in the left (53.35% vs 46.65% respectively). There was no significant effect of either, the side of ovulation, or age of the mare, on the side of EV fixation. However, EV fixation, was significantly (p < 0.001) more likely to occur in the right uterine horn in maiden and barren mares (65.75% vs 57.45% respectively). The age and reproductive status of the mare as well as foal heat breeding failed to demonstrate a consistent effect on pregnancy loss relative to the side of EV fixation. In lactating and foal heat bred mares, EVs were significantly (p < 0.0001) more frequently established in the contralateral horn to the one from which the mare delivered her most recent foal. In lactating mares, significantly (p < 0.05) higher embryonic and pregnancy losses were observed in the ipsilateral horn. In conclusion, (a) side of EV fixation was (i) independent of the side of ovulation and mare age (ii) significantly (p < 0.001) affected by reproductive status, (b) neither age of mare nor reproductive status had any effect on pregnancy loss rates relative to the side of EV fixation and (c) in lactating mares the EV had a greater chance of fixation and survival in the horn contralateral to the one from which the mare delivered her most recent foal.

Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus).

The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser horse stallion (stallion) and a jack. Semen was collected with an artificial vagina, at intervals of 48 to 72h (180 collections). The mean+/-SD (young [3.5 yr] vs. old [14 to 16 yr]) were Flehmen response frequency, 7.4+/-5.8 (8.1+/-3.0 vs. 7.0+/-2.0); number of mounts without erection, 1.1+/-1.3 (2.1+/-1.4 vs. 1.2+/-0.4, P<0.05); latency from first exposure to mare to full erection on the ejaculatory mount, 18.3+/-17.7min (25.3+/-21.3 vs. 12.2+/-6.2, P<0.05); latency from erection to insertion, 5.1+/-3.5sec (5.3+/-3.8 vs. 4.8+/-3.2); and duration of copulation from insertion to dismount after ejaculation, 25.4+/-7.8sec (22.1+/-2.9 vs. 28.1+/-9.3). In all jacks, sexual behavior was generally normal, with the notable absence of open mouth behavior. Mare estrous behavior was markedly less intense than that in the presence of a stallion and usually absent. Semen characteristics were gel free volume, 47.3+/-28.7mL; gel volume, 71.8+/-54.8mL; total motility, 84.3+/-6.0%; progressive motility, 74.3+/-74.5%; sperm vigor, 3.9+/-0.5 (scale 1 to 5); sperm concentration, 253x10(6) cells/mL; and total number of sperm, 10.3x10(9) cells. Copulation duration was significantly correlated with gel free volume (r=0.9) and gel volume (r=0.7). We concluded that (i) the sexual behavior of jacks during semen collection using mares was similar to that reported for natural mating to jennies, (ii) precopulatory and copulatory behavior for the young (less experienced) jacks and older (more experienced) jacks were generally similar (except number of mounts without erection and latency to full erection); (iii) semen obtained using mares as stimulus and mount females was similar to that reported with estrous jennies; and (iv) semen collection from previously conditioned jacks, using estrous mares, was appropriate for field collection of semen.

The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success.

Delayed uterine involution is a major cause of early reproductive failure in mares. Involution is affected by mare age, and foaling to covering interval. Involution rates vary between the previously non-gravid horn (PNGH), which recovers the quicker, and the previously gravid horn (PGH). Location of a pregnancy and its likely success may, therefore, be affected by its location relative to the previous pregnancy. This study aimed to determine: (i) the location of concepti in consecutive pregnancies; (ii) whether this varies with mare age or foaling to conception interval; (iii) whether location in relation to the previous pregnancy affects success. 1383 Thoroughbred mares were monitored by ultrasonic scanning during oestrus and early pregnancy. Significantly (p<0.01) more pregnancies were located in the PNGH (79.2%) than the PGH (20.8%). The number of pregnancies in PGH significantly increased with mare age (p<0.01) and foaling to conception interval (p<0.05). Significantly (p<0.001) more pregnancies located in the PGH (16.5%) failed, than those in the PNGH (4.6%). It can be concluded that most pregnancies locate in the PNGH where their chances of success are greatest. The larger number of pregnancies locating in the PGH in older mares and those with shorter foaling to conception intervals may in part account for the higher conceptus mortality rates in such mares. Hence breeding older mares on alternate years and maximising foaling to conception interval may improve reproductive success. Alternatively termination of pregnancies located in the PGH followed by timely recovering may be justifiable as might ET in older mares covered close to foaling.

Factors affecting gestation length in the Thoroughbred mare.

In order to assist in the accurate prediction of the timing of parturition in the mare true gestation length, along with the potential effect of a number of factors, was investigated. Data from 433 Thoroughbred foal pregnancies were used. Sequential ultrasonic scanning allowed the true gestation length (fertilisation-parturition) to be ascertained, as apposed to previous work, which used the mating-parturition interval. An average gestation length of 344.1 +/- 0.49 days was evident. Colt foal pregnancies were significantly (P < 0.001) longer (346.2 +/- 0.72) than fillies (342.4 +/- 0.65). Month of birth had a significant effect on gestation length in all foals (P < 0.001). With foals born in January having the shortest gestation lengths and those born in April the longest. Mare age, year of birth, stallion age, stud farm and the interval between ovulation and mating had no significant effect. It is concluded that (i) the gestation length range (315-388 days), all resulting in viable foals is noteworthy and of clinical importance when considering the classification of dysmaturity in foals, (ii) mares carrying colt foals due to be born in the middle of the breeding season (April) are likely to have the longer gestation lengths.

Effects of cool and hot humid environmental conditions on neuroendocrine responses of horses to treadmill exercise.

To determine the effects of exercise, high heat and humidity and acclimation on plasma adrenaline, noradrenaline, beta-endorphin and cortisol concentrations, five horses performed a competition exercise test (CET; designed to simulate the speed and endurance test of a three-day event) in cool dry (CD) (20 degrees C/40% RH) and hot humid (30 degrees C/80% RH) conditions before (pre-acclimation) and after (post-acclimation) a 15 day period of humid heat acclimation. Plasma adrenaline and noradrenaline concentrations pre-acclimation were significantly increased compared with exercise in the CD trial at the end of Phases C (P<0.05) and D (P<0.05 and P<0.01, respectively) and at 2 min recovery (P<0.01), with adrenaline concentrations still elevated after 5 min of recovery (P<0.001). Plasma beta-endorphin concentrations were increased at the end of Phases C (P<0.05) and X (P<0.01) and at 5 and 30 min recovery (P<0.05) in the pre-acclimation session. Plasma cortisol concentrations were elevated after the initial warm up period pre-acclimation (P<0.01) and at the end of Phase C (P<0.05), compared with the CD trial. A 15 day period of acclimation significantly increased plasma adrenaline concentrations at 2 min recovery (P<0.001) and plasma cortisol concentration at the end of Phase B (P<0.01) compared with pre-acclimation. Acclimation did not significantly influence noradrenaline or beta-endorphin responses to exercise, although there was a trend for plasma beta-endorphin to be lower at the end of Phases C and X and after 30 min recovery compared with pre-acclimation. Plasma adrenaline, noradrenaline, beta-endorphin and cortisol concentrations were increased by exercise in cool dry conditions and were further increased by the same exercise in hot humid conditions. Exercise responses post-acclimation suggest that adrenaline and noradrenaline may play a role in the adaptation of horses to thermal stress and that changes in plasma beta-endorphin concentrations could be used as a sensitive indicator of thermal tolerance before and after acclimation. The use of plasma cortisol as a specific indicator of heat stress and thermal tolerance before or after acclimation in exercising horses appears limited.

The effect of sodium heparin on equine articular cartilage.

This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no significant increase was found among the treatment groups (P =0.782). For gentamicin, no significant difference in percentage GAG loss was found between the control and three of the five treatment groups (P =0.667). The percentage GAG loss in the sodium heparin treated AC explants was greater than for any of the gentamicin-treated AC explants. It can be concluded that sodium heparin sulphate stimulates an increase in GAG release from equine articular cartilage explants, though no firm conclusions can be drawn on its use in treating equine infectious arthritis.

Ovulation rate and distribution in the thoroughbred mare, as determined by ultrasonic scanning: the effect of age.

Ovarian activity was monitored in 2181 oestrous cycles from 1136 thoroughbred brood mares to accurately determine overall ovulation rate, the relative distribution of ovulations between left and right ovary, whether mare age had an effect on these parameters and whether ovulation pattern in one cycle affected the pattern within the next. Ultrasonic scanning was used in preference to rectal palpation and slaughterhouse material due to its greater accuracy. Mare's ages ranged from 3 to 22 years. The results obtained indicated a similar incidence of ovulation occurring on the right (49.5% of all ovulations) or left ovary (50.5%). Multiple ovulations (MO) occurred in 22.4% of oestrous cycles. Significantly (P<0.01), more double ovulations (DO) were bilateral (57.2%) than unilateral (42.8%); 20.7% were unilateral right and 22.1% were unilateral left. There were 828 mares grouped into six age groups: A (3-5 years), B (6-8 years), C (9-11 years), D (12-14 years), E (15-18 years), F (18-22 years). The distribution of ovulation between the left and right ovary was not affected by age group, but significant association (P<0.01) was evident between age and MO. Group A had 15% of cycles resulting in MO compared to 19.4, 24.6, 25.8, 28.6 and 35.1% in groups B-F, respectively. A significant (P<0.01) association between MO in successive cycles was also demonstrated, with a greater chance (P<0.05) of successive single ovulations (SO) being on alternate ovaries. There was no significant repeatability of the classification of DO in one cycle compared to the next. We conclude that: (i) the mare is a symmetrical ovulator; (ii) 22.4% of cycles yield MO (ovulation rate 1.23); (iii) this MO incidence increased (P<0.01) with age; (iv) the exhibition of MO in one cycle was linked to an increased probability (P<0.01) of a MO occurring at the next one and (v) SO in one ovary was more likely (P<0.05) to be followed by a SO on the alternate ovary during the next cycle.

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.