Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing.

The cryotolerance of equine blastocysts larger than 300 µm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 µm, n = 14 and > 550 µm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 µm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 µm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.

Success rate in a clinical equine in vitro embryo production program.

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0-13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating.

Speed of in vitro embryo development affects the likelihood of foaling and the foal sex ratio.

The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.

Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos.

BACKGROUND: Monozygotic multiple pregnancy is rare in horses, but may be more common after transfer of an in vitro produced (IVP) embryo. OBJECTIVES: To determine the occurrence, incidence, characteristics and outcome of monozygotic siblings arising from in vivo and IVP equine embryos. STUDY DESIGN: Retrospective case series. METHODS: A total of 496 fresh in vivo and 410 frozen-thawed IVP blastocysts, produced by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes from Warmblood mares, were transferred into recipient mares. The likelihoods of pregnancy and multiple pregnancy were calculated, and the clinical features and outcome of any multiple pregnancy were recorded. RESULTS: The likelihood of pregnancy after transfer of a single IVP or in vivo embryo was 62% (254/410) and 83% (413/496) respectively. The incidence of multiple pregnancy was 1.6% (4/254) and 0% (0/413) for IVP and in vivo blastocysts, respectively. More specifically, three IVP blastocysts yielded twin embryo propers/fetuses, and one IVP conceptus developed three distinct embryonic bodies. Interestingly, only one embryonic vesicle was detected at all ultrasonographic examinations prior to embryo proper development. Multiple embryonic bodies only became apparent at later scans to check for an embryo proper and heartbeat, or when the recipient mare aborted. Two twin pregnancies aborted spontaneously at 3 and 9 months, respectively, while the heartbeat was lost from all three embryos in the triplet pregnancy before day 35 of gestation. Twin reduction by per rectum compression of one fetal thorax was attempted at day 50 of gestation in the fourth case; however, both fetuses were lost. MAIN LIMITATIONS: Small number of cases. CONCLUSIONS: In vitro embryo production resulted in a higher incidence of multiple monozygotic pregnancy, which could only be diagnosed after development of the embryo proper and is likely to result in pregnancy loss later in gestation if left untreated.

Factors affecting the likelihood of pregnancy and embryonic loss after transfer of cryopreserved in vitro produced equine embryos.

BACKGROUND: In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce. OBJECTIVES: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen-thawed IVP embryos and identify factors influencing success. STUDY DESIGN: Retrospective clinical study. METHODS: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The influence of donor mare (age, reproductive history), recipient mare (age, reproductive status, management; in-house vs. outpatient, day post-ovulation), embryo (interval from ICSI to blastocyst formation) and management factors (season when ovum pickup was performed, year and method of transfer) on likelihood of pregnancy and embryonic loss was examined, and the developmental stage of the IVP embryo at the time of transfer was estimated. RESULTS: The percentage of mares pregnant 7-10, 23 and 37 days after transfer was 56% (147/261), 49% (129/261), and 48% (124/261), respectively. Development of IVP embryos after transfer equated to day 5 or 6 in vivo embryos. With the exception of year of transfer, none of the factors had an impact on the likelihood of pregnancy or embryonic loss. Nevertheless, the likelihood of pregnancy tended to be lower for IVP embryos from infertile mares or when embryos were transferred into recipient mares on day 6 after ovulation rather than on day 4 or 5. Finally, the diameter of the embryonic vesicle 7 days post transfer was lower for pregnancies that were lost compared to those that were maintained. MAIN LIMITATIONS: Small sample size in some of the donor and recipient mare categories. CONCLUSIONS: Cryopreserved IVP embryos should be transferred into recipient mares on day 4 or 5 after ovulation and a slower rate of post transfer vesicle expansion indicates a higher risk of subsequent embryonic loss The Summary is available in Portuguese - see Supporting Information.

Advanced mare age impairs the ability of in vitro-matured oocytes to correctly align chromosomes on the metaphase plate.

BACKGROUND: Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. OBJECTIVES: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro-matured (IVM) oocytes. STUDY DESIGN: Morphometric and morphological analysis. METHODS: To investigate differences in spindle organisation and chromosome alignment between young and old mares, oocytes collected from slaughtered mares were divided into two groups depending on mare age (young, ≤14 years and old, ≥16 years), IVM and stained to visualise chromatin and alpha-tubulin. Spindle morphology, morphometry and chromosome (mis)alignment were evaluated by confocal microscopy and 3D image analysis. RESULTS: Oocytes from old mares showed a higher incidence of chromosome misalignment (47.4% vs. 4.5%; P<0.001) and a thicker metaphase plate (mean ± s.d.: 5.8 ± 1.0 µm vs. 4.9 ± 0.9 µm; P = 0.04) than oocytes from young mares. Although no differences in spindle morphometry were detected between old and young mares, an increased major spindle axis length was associated with chromosome misalignment (mean ± s.d.: 25.3 ± 6.1 µm vs. 20.8 ± 3.3 µm; P = 0.01) irrespective of age. MAIN LIMITATIONS: The oocytes were IVM and may not exactly reflect chromosome misalignment in vivo. CONCLUSIONS: Advanced mare age predisposes to chromosome misalignment on the metaphase II spindle of IVM oocytes. The compromised ability to correctly align chromosomes presumably predisposes to aneuploidy in resulting embryos and thereby contributes to the age-related decline in fertility and increased incidence of early pregnancy loss. The Summary is available in Portuguese - see Supporting Information.

Small day 8 equine embryos cannot be rescued by a less advanced recipient mare uterus.

Equine embryos tolerate an unusually large degree of negative uterine asynchrony (recipient mare up to 5 days behind the donor mare). By contrast, positive asynchrony of more than 2 days results in a high incidence of early embryonic loss (EEL). Day 8 embryos range in diameter from approximately 130-1300 µm, with embryos smaller than 300 µm reported to suffer an increased incidence of EEL. However, it is not known whether this reduced viability is due to intrinsically poor embryo quality, or to inadvertent recipient uterine stage-embryo (positive) asynchrony. To examine whether small embryos survive better in Day 4-5 recipients than in recipients with a more advanced uterine stage, the likelihood of pregnancy (PR) and EEL for 62 small (<300 µm) and 215 larger Day 8 horse embryos were compared after transfer to recipients at different uterine stages (Days 4-5, 6-7 and 8-9) using logistic regression. Overall, EEL was higher (21.2%; P < 0.05) for small than larger embryos (7.1%). However, neither PR nor EEL were influenced by the recipient's uterine stage at the time of transfer (P > 0.1). The EEL for small embryos transferred into Day 4-5, 6-7 and 8-9 recipients was 20.8, 18.7 and 25.0%, respectively. We conclude that embryos recovered on Day 8 with a diameter <300 µm are at increased risk of EEL due to reasons other than inadvertent positive asynchrony with the recipient mare's uterus.

Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares.

The diameter of embryos recovered from mares on Day 8 after ovulation varies greatly, from as little as 130 µm to as much as 2500 µm. Several factors have been proposed to affect embryo size at recovery, one of which is the type of semen (frozen vs fresh or cooled-transported) used to inseminate the mare. In addition, it has been shown that smaller embryos (<300 µm) recovered on Day 8 are less likely than larger embryos to result in successful pregnancy after transfer. However, whether the actual age of the embryo (interval from fertilization to flushing) in relation to its size also influences the post-transfer viability is unclear. The aims of this study were: a) To determine the effect of semen type (frozen-thawed vs cooled-transported) on embryo diameter after pre-ovulatory insemination; and b) To establish the relationship between embryonic age, embryo size and likelihood of pregnancy and pregnancy loss following transfer. A total of 179 embryos were recovered from mares inseminated with: frozen semen post-ovulation 8 days previously (G1; n = 35); cooled-transported semen pre-ovulation 8.5 days previously (G2; n = 95); frozen semen pre-ovulation 8.5 days previously (G3; n = 30); and frozen semen post-ovulation 9 days previously (G4; n = 19). The effect of embryonic age, type of semen, donor mare and its age, number of ovulations and embryos per flush on embryo diameter was tested using a general linear model of variance. In addition, the proportions and survivals of small embryos (<300 µm) in each group were compared with those of respective larger embryos by Fisher's exact test. Embryonic age (P < 0.001) and age of the donor mare (P = 0.07), but no other factor, influenced embryo diameter. The proportion of small embryos was 42.9, 10.5, 10.0 and 10.5% for Groups 1, 2, 3 and 4, respectively. The pregnancy status of recipient mares 35 days post-transfer for small embryos from Group 1 (12/15; 80.0%) was not different (P > 0.1) from that of recipients of small embryos from Groups 2 to 4 combined (8/15; 53.3%).

Likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus.

BACKGROUND: Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus. OBJECTIVES: To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET). STUDY DESIGN: Retrospective clinical study. METHODS: A total of 350 embryos recovered from 161 donor mares were transferred into 231 recipient mares during three consecutive breeding seasons. The following variables were analysed via two binary logistic regression models to determine their effect on pregnancy and EEL: 1) year of transfer, 2) season of transfer, 3) age of the recipient mare, 4) age of the donor mare, 5) operator performing the transfer, 6) singleton or twin embryo, 7) embryo size, 8) number of transfers to a given recipient in any one season, the use of 9) d-cloprostenol and 10) hCG in the recipient mare, 11) day of ovulation of the recipient mare at ET, 12) number of corpora lutea (CLs) at ET, and 13) duration of oestrus in the recipient mare. RESULTS: Age of the donor mare (P = 0.01), operator (P = 0.008), number of CLs at ET (P = 0.05) and the number of days of endometrial oedema during the oestrus preceding ET to the recipient mare (P = 0.004) influenced the likelihood of pregnancy. Early embryonic loss was influenced only by the year of transfer (P = 0.014). MAIN LIMITATIONS: Retrospective design of the study. The involvement of several veterinary surgeons over the 3-year period could have affected data recording. CONCLUSIONS: The likelihood of pregnancy in recipient mares is positively correlated with the duration of endometrial oedema during the oestrus preceding ET. This suggests a role for an adequate duration of oestrogenic priming during oestrus on uterine receptivity and embryo survival.

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years.

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.

Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation.

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na+/K+ gradient, which is dependent on an Na+-K+ antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

Relationship between colour flow Doppler sonographic assessment of corpus luteum activity and progesterone concentrations in mares after embryo transfer.

Colour-flow Doppler sonography has been described as a means of assessing corpus luteum (CL) function rapidly, because area of luteal blood vessels correlates well with circulating progesterone (P4) concentrations [P4] in oestrous cycling mares. The aim of this study was to assess the relationships between CL size and vascularity, and circulating [P4] during early pregnancy in mares, and to determine whether luteal blood flow was a useful aid for selecting an embryo transfer recipient. Equine embryos (n=48) were recovered 8 days after ovulation and were transferred to available recipient mares as part of a commercial program with the degree of synchrony in timing of recipient ovulation ranging from 1 day before to 4 days after the donor. Immediately prior to embryo transfer (ET), maximum CL cross-section and blood vessel areas were assessed sonographically, and jugular blood was collected to measure plasma [P4]. Sonographic measurements and jugular blood collection were repeated at day 4 after ET for all mares, and again at days 11, 18 and 25 after ET in mares that were pregnant. The number of grey-scale and colour pixels within the CL was subsequently quantified using ImageJ software. The CL blood flow correlated significantly but weakly with plasma [P4] on the day of transfer and on day 4 after ET in all mares, and on days 11 and 25 after ET in pregnant mares (r=0.30-0.36). The CL area and plasma [P4] were also correlated on each day until day 11 after ET (r=0.49-0.60). The CL colour pixel area decreased significantly after day 18, whereas CL area was already decreasing by day 4 after ET. The CL area, area of blood flow, or [P4] was predictive of pregnancy. Findings in the present study suggest that both CL area and blood flow are correlated with circulating [P4] at the time of transfer and in early pregnancy. Evaluation of the CL using B-mode or CF sonography, although practical, provides no improvement in the selection of recipients or prediction of pregnancy outcomes than methods employed currently.

Mare and foal survival and subsequent fertility of mares treated for uterine torsion.

REASONS FOR PERFORMING STUDY: Previous surveys have reported that mare and foal survival after correction of uterine torsion (UT) varies from 60 to 84% and from 30 to 54%, respectively. Furthermore, resolution via a standing flank laparotomy (SFL) has been associated with better foal, but not mare, survival. OBJECTIVES: To compare the success of SFL with other correction methods (e.g. midline or flank laparotomy under general anaesthesia; correction per vaginam). STUDY DESIGN: Retrospective analysis of clinical records. METHODS: Data on correction technique, stage of gestation, degree of rotation, survival and subsequent fertility for 189 mares treated for UT at 3 equine referral hospitals in The Netherlands during 1987-2007 were analysed. RESULTS: Mean stage of gestation at diagnosis was 283 days (range 153-369 days), with the majority of UTs (77.5%) occurring before Day 320 of gestation. After correction of UT, 90.5% of mares and 82.3% of foals survived to hospital discharge, between 3 and 39 days later, and to foaling. Multivariable logistic regression indicated that correction method and stage of gestation at UT affected survival of foals and mares. For foals, survival was 88.7% after SFL compared with 35.0% after other methods (P = 0.001). When UT occurred at <320 days, 90.6% of foals survived, compared with 56.1% at ≥320 days (P = 0.007). For mare survival, an interaction between stage of gestation and correction method was detected (P = 0.02), with higher survival after SFL (97.1%) than other methods (50.0%) at <320 days of gestation (P<0.01). When UT occurred at ≥320 days, mare survival did not differ between techniques (76.0 vs. 68.8%; P = 0.6). Of 123 mares that were bred again, 93.5% became pregnant; fertility did not differ between mares treated by SFL (93.9%) and other techniques (87.5%; P = 0.9). CONCLUSIONS: Standing flank laparotomy is the surgical technique of choice for resolving uncomplicated equine UT (i.e. with no coexisting gastrointestinal lesions) except when the stage of gestation exceeds 320 days.

Oral candidiasis following steroid therapy for oral lichen planus.

OBJECTIVES: The purpose of this multicentre study was to determine the incidence of oral candidiasis in patients treated with topical steroids for oral lichen planus (OLP) and to determine whether the application of a concurrent antifungal therapy prevented the development of an oral candidiasis in these patients. MATERIALS AND METHODS: Records of 315 patients with OLP seen at four Oral Medicine practices treated for at least 2 weeks with steroids with and without the use of an antifungal regimen were retrospectively reviewed. RESULTS: The overall incidence of oral fungal infection in those treated with steroid therapy for OLP was 13.6%. There was no statistically significant difference in the rate of oral candidiasis development in those treated with an antifungal regimen vs those not treated prophylactically (14.3% vs 12.6%) (P = 0.68). CONCLUSIONS: Despite the use of various regimens, none of the preventive antifungal strategies used in this study resulted in a significant difference in the rate of development of an oral candidiasis in patients with OLP treated with steroids.

Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion sperm.

Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm recovered from the caudae epididymides. Ejaculated samples were split, and one portion was centrifuged and re-suspended to reduce seminal plasma content. All samples were diluted to 50millionsperm/ml and divided into two portions, one of which was supplemented with 0.5mM d-penicillamine. After 48h, 96h, 144h and 192h storage, sperm motility was assessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining, and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillamine had no effect on motility of ejaculated sperm (P>0.05) but reduced total and progressive motility of epididymal sperm. Sperm chromatin integrity was not influenced by storage time, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercial semen extender was neither beneficial nor detrimental to the maintenance of quality in ejaculated semen stored at 5°C. The negative effect on motility of epididymal sperm may reflect differences in (membrane) physiology of spermatozoa that have not been exposed to seminal plasma.

Expression of leukaemia inhibitory factor at the conceptus?maternal interface during preimplantation development and in the endometrium during the oestrous cycle in the mare.

Leukaemia inhibitory factor (LIF) plays a critical role in blastocyst development and implantation in several species. The present study investigated mRNA and protein expression for LIF, as well as the low-affinity LIF receptor (LIFR) and interleukin-6 signal transducer (IL6ST), in equine endometrium, trophoblast and histotroph during early pregnancy and in the endometrium during the oestrous cycle. Endometrial LIF mRNA expression was upregulated after Day 21 of pregnancy, whereas LIF immunoreactivity increased in the endometrium on Day 28. Expression of LIF mRNA in the yolk sac membrane increased from Day 21 of pregnancy, whereas LIF immunoreactivity increased from Day 28 in the trophoblast. LIFR and IL6ST mRNA was expressed in the endometrium during both the oestrous cycle and early pregnancy and, although LIFR and IL6ST protein were localised to the glandular epithelium during the cycle and first 14 days of pregnancy, from Day 21 they were located in the luminal epithelium. Trophoblast expression of LIFR and IL6ST increased as pregnancy proceeded. In conclusion, LIF expression increased at the conceptus-maternal interface during capsule attenuation. Because contemporaneous upregulation of both LIFR and IL6ST was also observed in the trophoblast, we propose that LIF plays an important role in the development of endometrial receptivity for trophoblast growth, apposition and adhesion in mares.

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm.

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (-BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., -BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.