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Uterine pathologies are the most common causes of infertility in mares. This study aimed to establish an ex vivo blood-perfused model for equine uteri and investigate the possible effects of different cycle stages (estrus, diestrus and anestrus) on the applicability of the model. Uteri (n = 13) were collected at an abattoir, flushed with preservation solution, transported to the laboratory on ice, and isolated perfused with autologous blood for 6 h (n = 12). For negative control, one uterus was handled as described but left without perfusion for 6 h. The cycle stage was determined by examination of the ovaries for the presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Sonomicrometry crystals were implanted into the myometrium to record spontaneous contractions and the response to 0.5 IU oxytocin after 6 h of perfusion. Analyses of the arterial and venous perfusate were performed every hour to determine glucose consumption, lactate production, pH, lactate dehydrogenase activity (LDH), and potassium concentration (K

To successfully inseminate mares, precise detection of ovulation time is crucial, especially when using frozen-thawed semen. Monitoring body temperature, as has been described in women, could be a noninvasive way to detect ovulation. The objective of this study was to investigate the relationship between the time of ovulation and the variation of body temperature in mares based on automatic continuous measurements during estrus. The experimental group included 21 mares for 70 analyzed estrous cycles. When the mares showed estrous behavior, they were administered intramuscular deslorelin acetate (2.25 mg) in the evening. At the same time, monitoring of body temperature using a sensor device fixed at the left lateral thorax was started and continued for over 60 hours. In 2-hour intervals, transrectal ultrasonography was performed to detect ovulation. Estimated body temperature in the 6 hours following ovulation detection was on average 0.06°C +/- 0.05°C (mean +/- SD) significantly higher when compared with body temperature at the same time on the preceding day (P = .01). In addition, a significant effect of PGF2α administration for estrus induction on the body temperature was found, being significantly higher until 6 hours before ovulation compared to that of uninduced cycles (P = .005). In conclusion, changes in body temperature during estrus in mares were related to ovulation. The increase in body temperature immediately after ovulation might be used in the future to establish automatized and noninvasive systems to detect ovulation. However, the identified temperature rise is relatively small on average and hardly identifiable in the individual mares.

Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined size (i.e., ~20 μL) that were subsequently cooled by depositing on an aluminum alloy block placed in liquid nitrogen. Mathematical modeling was performed to compute the heat transfer and rate of cooling. The minimum CPA concentration needed for vitrification was determined for various CPAs (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide) and combinations thereof, while effects of droplet size and carrier solution were also identified. Sperm vitrification was eventually done using a glycerol/propylene glycol (1/1) mixture at a final concentration of 45% in buffered saline supplemented with 3% albumin and polyvinylpyrrolidon, while warming was done in standard diluent supplemented with 100 mM sucrose. The sperm concentration was found to greatly affect sperm membrane integrity after vitrification-and-warming, i.e., was found to be 21 ± 12% for 10 × 106 sperm mL-1 and 54 ± 8% for 1 × 106 sperm mL-1. However, an almost complete loss of sperm motility was observed. In conclusion, successful sperm vitrification requires establishing the narrow balance between droplet size, sperm concentration, CPA type and concentration, and exposure time.

In this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes.FTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in HPrincipal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage.CPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage.

The aim of this double-blinded placebo-controlled study was to investigate the effect of acetylsalicylic acid (ASA) on uterine blood flow, gestation length, placental and foal weights in pregnant mares. Sixteen Thoroughbred mares of different age (13.3 ± 4.1) and parity (7.4 ± 3.1) were randomly assigned to three treatment groups. Mares in group C (n = 4) served as controls and received 5,000 mg lactose orally once daily from D 120 (D 0 = day of ovulation) until parturition. Mares in group ASA1 (n = 7) received 5,000 mg ASA orally once daily from D 120 until parturition. Mares in group ASA2 (n = 5) received the same dose ASA as group ASA1 from D 120 to D 285, but twice daily from D 285 until parturition. Mares were examined by ultrasonography on D 14, 28, and 60, and in 21-days intervals from D 120 until parturition. The cross-sectional area, time average maximum velocity (TAMV), and pulsatility index were measured in both uterine arteries and the blood flow volume was calculated for each uterine artery and then summarized. All 16 mares carried a normal pregnancy and delivered live foals. In group ASA2 TAMV in the ipsilateral artery was significantly higher (P = .03) and these mares showed a tendency of increased total blood flow volume (P = .07) during late pregnancy (D 305-346). Results indicate that oral administration of 5,000 mg of ASA twice daily in pregnant mares causes a rise in uterine blood flow during late pregnancy.

There is an increasing demand for female fertility preservation. Cryopreservation of ovarian cortex tissue by means of vitrification can be done ad-hoc and for pre-pubertal individuals. Obtaining a homogeneous distribution of protective agents in tissues is one of the major hurdles for successful preservation. Therefore, to rationally design vitrification strategies for tissues, it is needed to determine permeation kinetics of cryoprotective agents; to ensure homogeneous distribution while minimizing exposure time and toxicity effects. In this study, Fourier transform infrared spectroscopy (FTIR) was used to monitor diffusion of different components into porcine ovarian cortex tissue. Water fluxes and permeation kinetics of dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG) were investigated. Diffusion coefficients derived from FTIR data, were corroborated with differential scanning calorimetry and osmometer measurements. FTIR allowed real-time spectral fingerprinting of tissue during loading with mixtures of protective agents, while discriminating between different components and water. Exposure to vitrification solutions was found to cause drastic initial weight losses, which could be correlated with spectral features. Use of heavy water allowed distinguishing water fluxes associated with dehydration and permeation, both of which were found to precede permeation of cryoprotective agents. Overall, DMSO and EG were found to permeate faster than GLY and PG. In mixtures, however, solutes behave differently. The non-invasive spectroscopic method described here to study permeation of vitrification solution components into ovarian tissue can be applied to many other types of engineered constructs, tissues, and possibly organs.

BACKGROUND Stallion-like or aggressive behaviour in mares affected by unilateral granulosa theca cell tumour (GTCT) is well-known, but use of a GnRH-vaccine as an alternative to surgical removal of the neoplastic ovary has not been investigated. OBJECTIVES To determine the effect of immunisation against GnRH on ovarian size, testosterone concentration, Anti-Mullerian hormone (AMH) concentration, and owner-reported behaviour in four mares affected by unilateral GTCT. STUDY DESIGN Retrospective case report. METHODS A presumptive diagnosis of GTCT was made in four mares based on clinical signs, behavioural changes, transrectal palpation, and ultrasonography. All mares were vaccinated twice with the GnRH-vaccine Improvac® on day 0 and on day 13-33. Further booster vaccinations were administered if aggressive behaviour recurred between days 15 and 498. Before and parallel to the vaccinations, serum levels of oestradiol, progesterone (P4), testosterone, and AMH were evaluated and transrectal ultrasonography was performed. RESULTS In all horses, analysis of serum levels of oestradiol, progesterone, testosterone, and AMH confirmed the clinical diagnosis of GTCT. Serum levels of testosterone dropped to baseline levels following the first two of three vaccination in all mares. In addition, AMH serum values decreased shortly after the second vaccination in three of four mares, and in one of the four mares returned to baseline levels. No further GTCT linked behaviour was reported by the owners and the affected ovaries diminished in size in all four cases. MAIN LIMITATIONS This report is a case series with a limited number of animals, no controls and no standardised immunisation protocol. CONCLUSIONS Repeated vaccinations with the GnRH-vaccine Improvac® mitigated owner-reported behavioural abnormalities and stopped tumour growth in four mares affected by unilateral GTCT over the entire observation period which extends to 7 years in one mare.

Anti-Mullerian Hormone (AMH) has been linked to reproductive tract abnormalities in mares and stallions. This study aimed at evaluating AMH as a biomarker for two reproductive conditions in mares. In the first part of this study, plasma AMH was evaluated as an early indicator of the onset of cyclicity in mares in the transitional period from the anovulatory phase during winter anoestrus to the cyclic phase during the breeding season. Ten mares between 8 and 17 years old were included in the experiment which lasted from mid-February until the end of April. Ovarian activity was monitored with ultrasonography three times per week, the detection of a corpus haemorrhagicum/luteum was documented and antral follicle counts (AFC) were recorded. Blood samples were collected weekly by jugular venipuncture during the whole study period to compare AMH concentrations before and after the first ovulation of the year. The second objective was to evaluate if plasma AMH concentrations in middle-aged mares are linked to fertility and could serve as a prognostic marker in that age group. A total of 41 privately-owned clinically sound mares aged between 12 and 21 years of various breeds were inseminated with fresh or frozen semen. Mares were scanned between day 14 and 20 and the “early pregnancy rate” included only positive pregnancy examinations after the first observed cycle in the season of each mare. Potential associations between the early pregnancy rate in the first cycle and the explanatory factors AMH concentrations, age, status of the mare, stud, development of post-breeding endometritis, number of inseminations and semen type were analysed using logistic regression models. In the first part of the study, correlation between AMH and AFC for the whole study period (P = 0.0002, ρ = 0.55) as well as prior to (P = 0.008, ρ = 0.58) and after the first ovulation (P = 0.0007, ρ = 0.69) were observed. However, AMH concentrations before and after the first ovulation of the year were not statistically different. The second part of the study revealed no association between early pregnancy rate and AMH concentrations or any of the other mentioned factors. In conclusion, this study showed no evidence of a difference between AMH concentrations before and after the first ovulation of the year thus not supporting the use of AMH as a biomarker to predict the onset of cyclicity in mares. We could furthermore not show a relationship between plasma AMH concentrations and early pregnancy rates in this cohort of animals.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.

Cellular membranes are exposed to extreme conditions during the processing steps involved in cryopreservation (and freeze-drying) of cells. The first processing step involves adding protective agents. Exposing cells to protective agents causes fluxes of both water and solutes (i.e., permeating cryoprotective agents) across the cellular membrane, resulting in cell volume changes and possibly osmotic stress. In addition, protective molecules may interact with lipids, which may lead to membrane structural changes and permeabilization. After loading with protective agents, subsequent freezing exposes cells to severe osmotic and mechanical stresses, caused by extra and/or intracellular ice formation and a drastically increased solute concentration in the unfrozen fraction. Furthermore, cellular membranes undergo thermotropic and lyotropic phase transitions during cooling and freezing, which drastically alter the membrane permeability and its barrier function. In this article, it is shown that membrane permeability to water and solutes is dependent on the temperature, medium osmolality, types of solutes present, cell hydration level, and absence or presence of ice. Freezing most drastically alters the membrane permeability barrier function, which is reflected as a change in the activation energy for water transport. In addition, membranes become temporarily leaky during freezing-induced fluid-to-gel membrane phase transitions, resulting in the uptake of impermeable solutes.

Transvaginal ultrasound-guided aspiration (TUA) is a procedure which can be used for the reduction of twins post-fixation in the mare. The aim of this study was to evaluate the effect of the age of mares and the day of gestation on the outcome of TUA treatment.In 88 mares, diagnosed pregnant with twins, TUA of the yolk sac or allantoic fluid was performed between day 30 and 62 of gestation. Mares were aged 3-22 years. Ultrasonographic examination for a viable singleton pregnancy was performed by referring veterinarians 5-7 days and 4 weeks after TUA. Based on reported findings, effects of age and day of gestation on pregnancy rates were evaluated.Four weeks after TUA, 67% of the cases resulted in a viable singleton pregnancy. Five to 7 days after TUA treatment, the success rate was 74%. The gestational period did not affect the outcome, irrespective of the age of the mare. In contrast, success rates decreased with increasing age of the mares (84% ≤ 7 years vs. 67% 8-14 years vs. 57% ≥ 15 years). In mares aged 8-14 years, a decrease in singleton pregnancies was observed, if TUA was performed after day 35 of gestation. Success rates were slightly higher, if twin vesicles were localized within separate uterine horns (73%) as compared to the same horn (66%). Differences in singleton pregnancy rates were not statistically significant (p0.05).TUA was found to be an effective procedure for reduction of twin pregnancies performed at days 30-62 of gestation. Success rates for singleton pregnancies were high for young mares ≤ 7 years old (84%) and middle aged mares treated before day 36 of pregnancy (74%). Duration of pregnancy at the time of TUA did not have a major impact on the outcome. Nevertheless, the procedure should optimally be performed around days 32-35 of pregnancy to allow for the possibility of natural reduction before treatment and rebreeding in case of a total pregnancy loss after TUA.Die transvaginale ultraschallgeleitete Aspiration (TUA) stellt eine Methode zur Zwillingsreduktion bei der Stute nach der Fixationsphase dar. Ziel der Studie war, den Einfluss von Alter und Trächtigkeitstag (TT) der Stuten auf die Resultate erfolgter TUAs zu beurteilen.Bei 88 Stuten im Alter von 3–22 Jahren mit diagnostizierter Zwillingsträchtigkeit wurde zwischen TT 30 und 62 eine Fruchtwasseraspiration vorgenommen. Die ultrasonographische Nach-untersuchung zur Feststellung einer intakten Einlingsgravidität führten die überweisenden Tierärzte 5–7 Tage sowie 4 Wochen nach der TUA durch. Basierend auf den mitgeteilten Befunden wurden die Einflüsse von Alter und Gestationstag der Stuten auf die Trächtigkeitsraten beurteilt.Die Rate für intakte Einlingsgraviditäten lag 4 Wochen nach der TUA bei 67%. Fünf bis 7 Tage nach dem Eingriff betrug die Erfolgsrate 74%. Unabhängig vom Alter der Stuten beeinflusste die Gestationsdauer die Ergebnisse nicht. Im Gegensatz dazu beeinträchtigte ein höheres Stutenalter die Behandlungserfolge (84% ≤ 7 Jahre vs. 67% 8–14 Jahre vs. 57% ≥ 15 Jahre). Bei 8–14 Jahre alten Stuten ergab sich eine geringere Reduktionsrate zur Einlingsgravidität, wenn die TUA nach dem 35. TT stattfand. Die Erfolgsrate für bicornuale Zwillingsträchtigkeiten (73%) lag leicht über der unicornualer (66%). Die Ergebnisse unterschieden sich nicht statistisch signifikant (p0,05).Die TUA erwies sich als effektive Maßnahme zur Zwillingsreduktion zwischen Tag 30 und 62 der Trächtigkeit. Hohe Raten von Einlingsgraviditäten wurden für junge Stuten ≤ 7 Jahre (84%) sowie vor dem 36. TT behandelte Stuten mittleren Alters (74%) erzielt. Die Trächtigkeitsdauer beeinflusste die TUA-Ergebnisse nicht maßgeblich. Dennoch sollte der Eingriff optimalerweise um TT 32–35 durchgeführt werden, um die Möglichkeit der natürlichen Zwillingsreduktion vor einer Behandlung sowie eine Wiederbelegung im Fall eines vollständigen Trächtigkeitsverlustes zu gewährleisten.

The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calcium chloride, and hardening (10 min). The first procedure resulted in structures with sperm residing in a liquid core surrounded by a spherical alginate shell, whereas the second procedure resulted in sperm embedded in solid beads of alginate matrix. It was found that use of calcium for alginate gelation resulted in decreased sperm motility as compared to using barium, and that encapsulation in solid beads had a negative impact on sperm plasma membrane intactness. Percentages of membrane intact sperm in barium-alginate core-shell structures were similar as found for ordinary diluted sperm, and did not change during 4 d storage at 5 °C. Sperm motility was reduced after direct recovery from core-shell structures, however, remained stable during 4 d storage leading to similar values as found for un-encapsulated sperm at this time point. Cryosurvival of sperm encapsulated in solid beads or core-shell structures was found to be lower compared to that of ordinary diluted sperm.

The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at −10°C in the absence of ice, and compared viability during supercooled storage with that during storage at 5°C. We found that addition of 2% Ficoll-400 to buffered saline and covering with mineral oil depressed the sample freezing point and inhibited surface-catalyzed nucleation. This allowed storage in a supercooled state at −10°C for up to 7 d. Supplementing specimens with sperm, however, increased the incidence of sample freezing. Nonetheless, with 50×106 sperm mL−1, about 40% of the samples turned out to be non-frozen. Adding 100 mM sucrose was found to preserve sperm membrane intactness during supercooled storage, although this resulted in lower percentages as found with refrigerated storage. Sperm motility appeared to be lost during supercooled storage but could be partly restored by substituting buffered saline with a milk-based extender as base medium. Percentages of membrane intact sperm, however, were found to be lower. Supercooled storage holds promise for semen preservation, but further optimization of the storage solution is required to preserve sperm motility.

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6M concentrations compared to only 1M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.

Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions. Stallion sperm was freeze-dried in various formulations consisting of reducing or nonreducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 to 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9-11% and 33-52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.

A 23-year-old Warmblood stallion with pituitary pars intermedia dysfunction (PPID) was treated with 0.002 mg/kg body weight pergolide orally once a day, starting in July. Semen collections during 5 consecutive days each were performed before starting and at 90 days after initiation of the therapy in order to assess the effect of the therapy on semen volume, concentration, total sperm count, motility, and viability. The only changes observed were lowered semen volume compensated by increased semen density resulting in an equal total sperm count. Possible causes for these observations include altered prolactin levels, the influence of season, or pergolide acting directly on the ejaculation process. This case report provides first evidence that pergolide therapy in PPID may alter semen parameters, but these changes are unlikely to affect fertility. However, this observation in a single individual must be confirmed with experiments on multiple stallions.

In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min−1. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cooling rates for cryopreservation. Sperm cryosurvival was experimentally determined through flow cytometric assessments on membrane intactness and using computer-assisted analysis of motility. Sperm could withstand exposure to 1500 mM concentrations prefreeze for all CPAs tested. The overall highest cryosurvival rates were obtained with DMF, followed by GLY and EG, whereas the use of PG and DMSO resulted in poor cryosurvi...

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

The present study has the objectives to analyse effects influencing the gestation length (GL) and the parturition conception interval (PCI) in Hanoverian warmblood horses and subsequently to estimate heritabilities for the GL and the PCI. Data comprised artificial insemination records of the years 2008–2014 from the National State Stud Celle, Germany, and corresponding foaling data from the Hanoverian Breeding Society, Germany. A total of 19,315 seasonal records including 10,294 mares and 276 stallions were available for GL and 10,244 records for PCI with 5881 mares and 234 stallions. The average GL was 341.7±10.7 days. The fixed effects of the year and month of insemination, the age of the mare at the time of insemination, whether the mare had a foal with her from the previous season, the sex of the foal and the random effects of the mare and the stallion had a significant effect on the GL. For the effect of the month of insemination the GL was longest in the months of December to February and shortest in July and August. It was shortest in 2–4-year old mares and longest in mares >19 years. Also GLs were longer if the mare did not have a foal at foot from the previous season than in mares with a foal with her. Pregnancies with colts were longer than those with fillies. The average PCI was 38.1±25.9 days. Year and month of insemination, the age of the mare at the time of insemination and the mare as well as the stallion had a significant effect on the PCI. PCI was shortest in the months of December to February and increased until July and August. The PCI was longest in the youngest and the oldest age class of the mares. Heritabilities were estimated in linear animal models. A model including the maternal and paternal genetic effect resulted in heritabilities of 0.234 for the mare and 0.024 for the stallion for GL and 0.026 and 0.013 respectively for the PCI. Heritabilities for GL in a model including the maternal and direct genetic effects were 0.269 for the mare and 0.066 for the embryo. Mare-related effects seem to have the largest impact on GL, whereas environmental effects like a good post-parturition management highly influence the PCI.

The major histocompatibility complex (MHC) influences sexual selection in various vertebrates. Recently, MHC-linked social signaling was also shown to influence female fertility in horses (Equus caballus) diagnosed 17 days after fertilization. However, it remained unclear at which stage the pregnancy was terminated. Here we test if MHC-linked cryptic female choice in horses happens during the first days of pregnancy, i.e., until shortly after embryonic entrance into the uterus and before fixation in the endometrium. We exposed estrous mares to one of several unrelated stallions, instrumentally inseminated them with semen of another stallion, and flushed the uterus 8 days later to test for the presence of embryos. In total 68 embryos could be collected from 97 experimental trials. This success rate of 70.1% was significantly different from the mean pregnancy rate of 45.7% observed 17 days after fertilization using the same experimental protocol but without embryo flushing. Embryo recovery rate was not significantly dependent on whether the mares had been socially exposed to an MHC-dissimilar or an MHC-similar stallion. These observations suggest that MHC-linked maternal strategies affect embryo survival mainly (or only) during the time of fixation in the uterus.

Background Warmblood fragile foal syndrome (WFFS) is a lethal condition detected in Warmblood horses. Its origin and association with performance traits and fertility among horse populations is unknown. Objectives To validate the previously identified WFFS type 1 (WFFST1)-associated missense variant PLOD1:c.2032G>A and to investigate its distribution among various horses with particular focus on Hanoverian breed, as well as its pathomorphological picture. The study aimed at identifying the origin of the mutant allele and its correlation with performance and fertility traits in Warmblood horses. Study design Retrospective case-control and association study. Methods WFFST1 variant was validated using whole genome sequencing (WGS) in 78 equids. In an affected foal with a homozygous mutant genotype, necropsy was performed. Skin samples were examined using histology and transmission electron microscopy. Pathway analysis was performed to trace back 81 genetic carriers to the most common recent ancestor. Furthermore, generalised linear model analysis was employed to test estimated breeding values (EBVs) for differences in performance and fertility traits among different genotypes in Hanoverian horses. Results WFFST1 variant had the lowest minor allele frequency among all variants detected in WGS data in the region of PLOD1. Further genotyping of this variant revealed allele frequencies of 0.14 in Hanoverian horses. Histological investigations of the WFFST1-affected foal showed loosely arranged collagen fibres in the dermis. Ultrastructurally, multifocal areas with degraded collagen fibrils and fibrillar plaques were detected. Further pathway analysis revealed a stallion from the Hanoverian sire F/W line as the most common recent ancestor of all tested genetic carriers. Furthermore, WFFST1 variant was found to be correlated with EBVs for gait-related traits as well as conformation and dressage. Main limitations Study evaluated carriers and cases only from Europe. Conclusions This study provides a comprehensive evaluation of WFFST1 variant and traces it back to its potential origin.

Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ± standard deviation = 9.5 ± 4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were evaluated immediately, 24 and 48 h after cooled storage at 5 °C. At the two storage time points, membrane lipid peroxidation was determined using the BODIPY-C11. Additionally, blood samples were collected at Weeks 0, 1, 5 and 9, and analysed for antioxidant status, consisting of superoxide dismutase, cholesterol, thiobarbituric acid reactive substances, and non-esterified fatty acids. Due to the nature of the data, the Mann-Whitney U test was applied as preliminary analysis. The BODIPY-C11 in the semen was less at 24 h and greater at 48 h after collections in Week 1 to 3 (P < 0.01) and Week 1 to 10 (P

Objectives of the present study were to estimate genetic parameters for frozen-thawed semen traits of 271 fertile German Warmblood stallions and genetic correlations with raw semen quality traits. Semen samples were collected from stallions utilized for semen collection and artificial insemination (AI) on the Lower Saxon National Stud Celle and the North Rhine-Westphalian National Stud Warendorf. Semen quality variables were analyzed in 63,972 raw (gel-free volume, concentration, progressive motility, number of sperm) and 3681 frozen-thawed samples (motility, DNA fragmentation index (DFI), non-viable sperm). A multivariate linear animal model was used to estimate additive genetic and permanent environmental variances among stallions as well as estimated breeding values (EBVs) for all semen traits. Heritability estimates were greatest for DFI (h2 = 0.45) and least for non-viable sperm counts ( h2 = 0.11). Additive genetic correlations between progressive sperm motility in raw semen and DFI (rg = −0.79) as well as non-viable sperm (rg = −0.45) were negative. The EBVs for frozen-thawed semen traits ranged from 49 to 181 with mean reliabilities of 0.28 to 0.43. The EBVs for progressively motile sperm post-thawing and DFI were the most highly correlated traits with EBVs for stallion fertility (r = 0.38 and r = −0.17). Stallions with relatively greater EBVs for progressive motility in raw semen may be most suitable when freezing semen for storage and subsequently thawing it for AI. Using EBVs for semen traits in selection of stallions to AI mares appears as an option for genetic improvement to enhance fertility after AI.

The objectives of the present study were to analyze systematic effects on semen quality traits from fresh and frozen-thawed semen collected in and outside season. A total of 4,681 reports on semen traits of 121 stallions representing Arabian, Thoroughbred, Quarter Horse and four warmblood breeds used for artificial insemination at the Lower Saxon National stud Celle were edited for analysis of gel-free volume, sperm concentration, total number of sperm and total number of motile sperm in fresh semen and in frozen-thawed semen progressive motility, DNA fragmentation index and non-viable sperm. Month, year, age, breed and stallions effects were analyzed with a linear mixed model procedure. Breed differences were significant for sperm concentration, total number of sperm, total motile sperm number and DNA fragmentation index. Hanoverian stallions showed significant higher least squares means for sperm concentration, total number of sperm and total motile sperm number in fresh semen, whereas Thoroughbred had significant higher least squares means for DNA fragmentation index. Stallions with an age of 2-6 years had significant lower least squares means in sperm concentration, total number of sperm and total motile sperm number and progressively motile sperm post-thawing than the other age groups. Month was significant for all semen traits but progressively motile sperm post-thawing. Month by age class interaction showed a significant influence. Inter-stallion variance accounted for 27-71% and the two-way stallion by month interaction for 2-7% of the total variance of semen quality traits. The largest proportion of inter-individual variance among stallions was obtained for the DNA fragmentation index.

Many years of poor results of equine embryo cryopreservation has produced a lack of confidence in this technique. Embryo cryopreservation has been successfully used for more than 20 years in other species like bovine and human. The large size of the embryos and the presence of a capsule impermeable to cryoprotectants have been the two main reasons for the failure. In the last few years, a mayor breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after breaching the capsule and collapsing the blastocoel cavity. In the present study, we compared the pregnancy rates obtained by vitrification or cryopreservation by slow freezing of embryos smaller than 300 μm. No difference was found between vitrification and slow freezing of embryos