Cooling dynamics of droplets exposed to solid surface freezing and vitrification.

Solid surface freezing or vitrification (SSF/SSV) can be done by depositing droplets of a sample, e.g., cells in a preservation solution, onto a pre-cooled metal surface. It is used to achieve higher cooling rates and concomitant higher cryosurvival rates compared to immersion of samples into liquid nitrogen. In this study, numerical simulations of SSF/SSV were conducted by modeling the cooling dynamics of droplets of cryoprotective agent (CPA) solutions. It was assumed that deposited droplets attain a cylindrical bottom part and half-ellipsoidal shaped upper part. Material properties for heat transfer simulations including density, heat capacity and thermal conductivity were obtained from the literature and extrapolated using polynomial fitting. The impact of CPA type, i.e., glycerol (GLY) and dimethyl sulfoxide (DMSO), CPA concentration, and droplet size on the cooling dynamics was simulated at different CPA mass fractions at temperatures ranging from -196 to 25 °C. Simulations show that glycerol solutions cool faster compared to DMSO solutions, and cooling rates increase with decreasing CPA concentration. However, we note that material property data for GLY and DMSO solutions were obtained in different temperature and concentration ranges under different conditions, which complicated making an accurate comparison. Experimental studies show that samples that freeze have a delayed cooling response early on, whereas equilibration times are similar compared to samples that vitrify. Finally, as proof of concept, droplets of human red blood cells (RBCs) were cryopreserved using SSV/SSF comparing the effect of GLY and DMSO on cryopreservation outcome. At 20% (w/w), similar hemolysis rates were found for GLY and DMSO, whereas at 40%, GLY outperformed DMSO.

Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

In the past, most research in equine reproduction has been performed in vivo but the use of in vitro and ex vivo models has recently increased. This study aimed to evaluate the functional stability of an ex vivo hemoperfused model for equine uteri with molecular characterization of marker genes and their proteins. In addition, the study validated the respective protein expression and the aptness of the software QuPath for identifying and scoring immunohistochemically stained equine endometrium. After collection, uteri (n = 12) were flushed with preservation solution, transported to the laboratory on ice, and perfused with autologous blood for 6 h. Cycle stage was determined by examination of the ovaries for presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Samples were obtained directly after slaughter, after transportation, and during perfusion (240, 300, 360 min). mRNA expression levels of progesterone (PGR), estrogen (ESR1) and oxytocin (OXTR) receptor as well as of MKI67 (marker of cell growth) and CASP3 (marker of apoptosis) were analyzed by RT-qPCR, and correlation to protein abundance was validated by immunohistochemical staining. Endometrial samples were analyzed by visual and computer-assisted evaluation of stained antigens via QuPath. For PGR, effects of the perfusion and cycle stage on expression were found (P < 0.05), while ESR1 was affected only by cycle stage (P < 0.05) and OXTR was unaffected by perfusion and cycle stage. MKI67 was lower after 360 min of perfusion as compared to samples collected before perfusion (P < 0.05). For CASP3, differences in gene expression were found after transport and samples taken after 240 min (P < 0.05). Immunohistochemical staining revealed effects of perfusion on stromal and glandular cells for steroid hormone receptors, but not for Ki-67 and active Caspase 3. OXTR was visualized in all layers of the endometrium and was unaffected by perfusion. Comparison of QuPath and visual analysis resulted in similar results. For most cell types and stained antigens, the correlation coefficient was r > 0.5. In conclusion, the isolated hemoperfused model of the equine uterus was successfully validated at the molecular level, demonstrating stability of key marker gene expression. The utility of computer-assisted immunohistochemical analysis of equine endometrial samples was also confirmed.

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

BACKGROUND: Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. OBJECTIVES: To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. STUDY DESIGN: Retrospective case series. METHODS: Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. RESULTS: TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. MAIN LIMITATIONS: Missing retrospective data despite extensive follow-up. CONCLUSION: This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

Preserving frozen stallion sperm on dry ice using polymers that modulate ice crystalization kinetics.

Cryopreserved semen is routinely shipped in liquid nitrogen. Dry ice could serve as an alternative coolant, however, frozen storage above liquid nitrogen temperatures (LN2, -196 °C) may negatively affect shelf-life and cryosurvival. In this study, we determined critical temperatures for storage of cryopreserved stallion sperm. We evaluated: (i) effects of cooling samples to different subzero temperatures (-10 °C to -80 °C) prior to storing in LN2, (ii) stability at different storage temperatures (i.e., in LN2, dry ice, -80 °C and -20 °C freezers, 5 °C refrigerator), and (iii) sperm cryosurvival during storage on dry ice (i.e., when kept below -70 °C and during warming). Furthermore, (iv) we analyzed if addition of synthetic polymers (PVP-40, Ficoll-70) modulates ice crystallization kinetics and improves stability of cryopreserved specimens. Sperm motility and membrane intactness were taken as measures of cryosurvival, and an artificial insemination trial was performed to confirm fertilizing capacity. We found that adding PVP-40 or Ficoll-70 to formulations containing glycerol reduced ice crystal sizes and growth during annealing. Post-thaw sperm viability data indicated that samples need to be cooled below -40 °C before they can be safely plunged and stored in LN2. No negative effects of relocating specimens from dry ice to LN2 and vice versa became apparent. However, sample warming above -50 °C during transport in dry ice should be avoided to ensure preservation of viability and fertility. Moreover, addition of PVP-40 or Ficoll-70 was found to increase sperm cryosurvival, especially under non-ideal storage conditions where ice recrystallization may occur.

Success of different therapies for bacterial endometritis in stud farm practice.

Bacterial endometritis is a major problem in equine reproduction usually treated with antibiotics, however reports of success rates are scarce. This study collected data from mares diagnosed with intrauterine bacterial growth and compared the outcome of different therapies for bacterial endometritis in German stud farm practice. Data on mares with positive uterine culture results were collected retrospectively in veterinary practices (n = 5; 2018-2022). Information relating to 30 factors (mare, diagnostics, therapy, pregnancy rate) of bacterial endometritis cases (n = 772) were recorded and analyzed. Possible effects on treatment success (positive pregnancy result in the first cycle after treatment) were tested by binomial logistic regression. In most cases ß-hemolytic streptococci were detected (n = 707). Treatments for the endometritis included trimethoprim-sulfonamides (n = 409), procaine-penicillin (n = 227), marbofloxacin (n = 53) or no antibiotics (n = 59) and most antibiotics were administered systemically (n = 711) rather than locally (n = 23). Uterine lavage was reported in 49 % of mares. Uterotonic drugs were administered in 42.2 % of mares. Breeding programs included artificial insemination (AI) with chilled semen (n = 667), AI with frozen semen (n = 169), transfer of fresh (n = 112) or cryopreserved (n = 27) embryos and natural cover (n = 27). In the first cycle after treatment, the pregnancy rate was 47 % and it rose to 69 % by end of the season. Treatment success was affected by duration of antibiotic treatment, veterinary practice, and presence of clinical signs. In conclusion, reported treatment practices in German stud farm practice resulted in acceptable pregnancy results and the multiple binomial logistic regression approach identified factors affecting the pregnancy outcome in this dataset.

Frequency of potentially pathogenic bacterial and fungal isolates among 28,887 endometrial samples from mares, with an emphasis on multi-drug resistant bacteria in Germany (2018-2022).

Antimicrobial-resistant bacteria pose a serious threat to the wellbeing of animals and humans. In equine reproduction, endometritis caused by facultative microbial pathogens is a condition, which is usually treated with antibiotics. Data from Germany on prevalence of facultative pathogenic microorganisms cultured in samples from the equine uterus and the frequency of multi-drug resistant (MDR) bacteria is lacking. The aim of the study was to provide representative numbers for both. Microbiological culture results (n = 28,887) of endometrial samples submitted to a large veterinary diagnostic laboratory from 2018-2022 were analyzed. An average of 25.9 % of the culture results showed growth of facultative pathogenic bacteria. The dominant isolated bacteria were ß-hemolytic streptococci (79.7 %) followed by Escherichia (E.) coli variatio haemolytica (5.2 %). E. coli were cultured in 4.3 % of the samples and occurred more often than Klebsiella pneumoniae (3.9 %), Candida species (2.9 %), Pseudomonas aeruginosa (2.0 %), and Staphylococcus aureus (1.5 %). Antibiotic susceptibility testing revealed sensitivity of ß-hemolytic streptococci towards penicillins in almost 100 % of the cultured samples (99.5 %). E. coli-isolates were sensitive to gentamicin in 96.2 % of the cases. The frequency of multidrug-resistant extended spectrum beta-lactamase (ESBL)-positive bacteria and methicillin-resistant Staphylococcus aureus (MRSA) was 3.1 % of all positive culture results. The number of ESBL-positive isolates (n = 159) and MRSA was stable from 2018-2022. In conclusion, the situation regarding occurrence of MDR bacteria in Germany is favorable, but should further be monitored.

Age and treatment on the day of embryo transfer in recipient mares affect likelihood of pregnancy.

OBJECTIVE: This retrospective evaluation of data from a large commercial embryo transfer facility aimed to determine the extent to which age and treatment on the day of embryo transfer in recipient mares influence the likelihood of pregnancy. MATERIAL AND METHODS: Embryo recovery was carried out on days 8-10 post-ovulation using transcervical uterine flushing. Recipient mares grouped according to their age were treated once on the day of embryo transfer (Day 3-8 post ovulation) and were assigned randomly to 1 of 3 groups: Mares in Group A (n=101) received antispasmodic, antimicrobial, and anti-inflammatory drugs. Mares in Group B (n=100) received gentamicin and flunixin meglumine. Group C (control) (n=103) did not receive any treatment. Detomidine (0.008 mg/kg bwt i.v.) was administered to all recipients before transfer of the embryo. The influence of treatment and recipient´s age was calculated using binary logistic regression. RESULTS: Day 16 post-transfer pregnancy rates were highest in Group A (74/101, 73.3a%), when compared to Group B (60/100, 60%), and Group C (57/103, 55.3b%) (a vs b, p<0.05). Pregnancy loss rates at D45 were not different between groups, A (8/74, 10.8%), B (5/60, 8.3%), and C (6/57, 10.5%), respectively (p>0.05). Pregnancy losses were increased in recipient mares 17-22 years (33.3a%) compared to younger recipient mares (2-6 years 7b%, 7-11 years 10%, 12-16 years 8b%) (a:b p<0.05). The regression model showed that the predicted probability for pregnancy after embryo transfer decreased as the age of the recipient mare increased for treated recipients in Group A (p=0.012), there was no effect of treatment and recipient´s age in Group B, and a decreased likelihood of pregnancy in recipients of advanced age (≥12 years of age) in untreated recipients (group C). CONCLUSIONS AND CLINICAL RELEVANCE: Likelihood of pregnancy increased following single administration of antispasmodic, antimicrobial, and anti-inflammatory drugs at the time of embryo transfer in recipients 2-12 years of age. Likelihood of pregnancy in recipients decreased in recipients≥12 years of age. These results, obtained under the conditions of a large commercial embryo transfer program, offer an opportunity to improve pregnancy rates in recipient mares≤12 years of age.

Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.

To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4-positive cells [0.015 ± 0.02%]), whereas CD21-positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant-treated ejaculates can be used as an additional tool for spermatological examination in stallions.

Increase of Body Temperature Immediately After Ovulation in Mares.

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.

Comparison of systemic trimethoprim-sulfadimethoxine treatment and intrauterine ozone application as possible therapies for bacterial endometritis in equine practice.

Bacterial endometritis is one of the major problems in equine reproduction and usually treated with antimicrobial drugs. The study aimed to compare the effects of intrauterine ozone application and systemic antibiotic treatment (trimethoprim-sulfadimethoxine) on intrauterine bacterial growth and possible side effects on the endometrium in a clinical setting. Mares (n = 30) with signs of endometritis (positive uterine bacterial culture and cytological findings) were assigned randomly to different treatments: intrauterine insufflation of an ozone-air-mix (240 ml, 80 µg ozone/ml) twice at a 48 h-interval (Ozone; n = 10), systemic antibiotic therapy with trimethoprim-sulfadimethoxine (30 mg/kg, p.o., twice daily) for 5 days (TMS; n = 10), or intrauterine insufflation of air (240 ml, sterile-filtered) twice at a 48 h-interval (air; n = 10). Endometrial biopsy for histological examination was obtained before the treatment. Histological examination revealed no differences among groups. A control examination, including transrectal ultrasound, bacterial culture, cytological evaluation, and biopsy, was performed 7 days after the last treatment. Overall bacterial growth was reduced in every group after the treatment (p < 0.05), irrespective of the therapy [Ozone: 4/9 (positive culture after treatment/number of mares), TMS: 3/10 and Air: 6/10; p > 0.05]. However, Ozone and TMS (p < 0.05) were more effective in reducing growth of gram-negative bacteria as compared to Air (p > 0.05). No effects on the number of polymorphonuclear granulocytes (cytology) were observed (p > 0.05). In conclusion, trimethoprim-sulfadimethoxine and intrauterine ozone insufflation are safe treatment options for bacterial endometritis in mares but the efficacy of both treatments in reducing bacterial growth did not result in a complete absence of intrauterine bacterial growth.

Infrared spectroscopic analysis of hydrogen-bonding interactions in cryopreservation solutions.

BACKGROUND: 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. METHODS: FTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H2O and D2O, and their thermal response was analyzed using van 't Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye. RESULTS: Principal 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. CONCLUSIONS: 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.

Effect of Acetylsalicylic Acid on Uterine Blood Flow, Gestation Length, Foal Birth Weight and Placental Weight in Pregnant Thoroughbred Mares - A Clinical Pilot Study.

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.

Increase of skin temperature prior to parturition in mares.

Prediction of impending foaling is highly desirable as early intervention may improve mare and foal outcomes. However, monitoring the peripartum mare is a time-consuming challenge for breeders and many foaling prediction systems have limitations. "Heating up" of the mare is empirically used by breeders as a sign of upcoming parturition in mares. The purpose of this study was to investigate if an increase in skin temperature shortly before parturition is detectable and to determine whether such physiological changes could be an additional valuable parameter to predict foaling. For that, 56 foalings of 14 Warmblood mares, 5 Arabian mares, 27 Thoroughbred mares, and 2 mares of other breeds were analyzed in this 2-year-study. Eight mares were monitored in both years. Mares were between 4 and 22 years old (average: 10 ± 5.5 years) and the mean pregnancy length was 342 days (±9 days), resulting in 14 births from primiparous mares and 42 multiparous mares. For monitoring the periparturient mares, the Piavet® system (Piavita AG, Zurich, Switzerland) was fixed daily when the mares had returned from the field between 4:00 and 6:00 p.m. and collected the next morning between 6:30 and 7:30 a.m. until the time of foaling. Nocturnal rhythms of the skin temperature with the highest values at the start of measurements and a nadir at 6:00 a.m. were observed. On the foaling night, we found a rise in skin temperature starting on average around 90 min prepartum. Skin temperatures recorded at 50 min before parturition and at each 5 min time point until rupture of the allantochorion were significantly higher (p < 0.05) than the mean temperatures measured in the 5 nights before parturition at the same time, reaching a difference of approximately 0.5 °C. There was a significant effect of parity (p = 0.04) on skin temperature during the last hours before foaling where primiparous mares showed a higher mean temperature than uni- or pluriparous mares as early as from 180 min on before parturition. In conclusion, our study shows an increase in skin temperature in most mares within 90 min before birth. Using new biomechanical and digital technologies, this finding could generate an additional potential parameter for the detection of impending parturition. However, skin temperature cannot be used as the only predictive diagnostic of impending parturition in the absence of other parameters.

Evaluation of an ex vivo model of the blood-perfused equine uterus.

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+). Biopsy samples were obtained directly after slaughter, after transportation, after equilibration, after 4, 5, and 6 h of perfusion, and immediately after removal from the perfusion system. The uteri's glucose consumption and lactate production increased over time (p < 0.05), but no differences among cycle stages were detected. pH (arterial and venous) increased over time (p < 0.05). No changes for LDH were observed. K+ increased after 4 h of perfusion (p < 0.05), but was unaffected by the cycle stage. Spontaneous contractions were present in all perfused uteri, but myometrial activity in the negative control was limited to the 2nd hour of perfusion. No effects of cycle stage on contraction amplitude and duration after oxytocin administration were detected. The cycle stage did not affect frequency (except after 5 h of perfusion), amplitude, duration of contractions, or edema formation. Histology revealed congestion of endometrial capillaries after 4, 5, and 6 h perfusion time. In conclusion, the ex vivo model was capable of supporting the functionality of equine uteri for 6 h. However, viability and histomorphology of the endometrium appeared to be impaired after 4 h of perfusion. Effects of the cycle stage on the applicability of the model were absent.

Alginate encapsulation of stallion sperm for increasing storage stability.

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.

Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice.

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 days. 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.

Loading equine oocytes with cryoprotective agents captured with a finite element method model.

Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane was used to simulate oocyte volume responses and concomitant intracellular CPA concentrations during the exposure of oocytes to CPA solutions. The results of the analytical simulations were subsequently used to develop a phenomenological finite element method (FEM) continuum model to capture the response of oocytes exposed to CPA solutions with spatial information. FEM simulations were used to depict spatial differences in CPA concentration during CPA permeation, namely at locations near the membrane surface and towards the middle of the cell, and to capture corresponding changes in deformation and hydrostatic pressure. FEM simulations of the multiple processes occurring during CPA loading of oocytes are a valuable tool to increase our understanding of the mechanisms underlying cryopreservation outcome.

Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations.

Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

Fourier transform infrared spectroscopy coupled with machine learning classification for identification of oxidative damage in freeze-dried heart valves.

Freeze-drying can be used to ensure off-the-shelf availability of decellularized heart valves for cardiovascular surgery. In this study, decellularized porcine aortic heart valves were analyzed by nitroblue tetrazolium (NBT) staining and Fourier transform infrared spectroscopy (FTIR) to identify oxidative damage during freeze-drying and subsequent storage as well as after treatment with H2O2 and FeCl3. NBT staining revealed that sucrose at a concentration of at least 40% (w/v) is needed to prevent oxidative damage during freeze-drying. Dried specimens that were stored at 4 °C depict little to no oxidative damage during storage for up to 2 months. FTIR analysis shows that fresh control, freeze-dried and stored heart valve specimens cannot be distinguished from one another, whereas H2O2- and FeCl3-treated samples could be distinguished in some tissue section. A feed forward artificial neural network model could accurately classify H2O2 and FeCl3 treated samples. However, fresh control, freeze-dried and stored samples could not be distinguished from one another, which implies that these groups are very similar in terms of their biomolecular fingerprints. Taken together, we conclude that sucrose can minimize oxidative damage caused by freeze-drying, and that subsequent dried storage has little effects on the overall biochemical composition of heart valve scaffolds.

high-throughput droplet vitrification of stallion sperm using permeating cryoprotective agents.

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.