Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols.

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h.

Advances in Stallion Semen Cryopreservation.

The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation and consequently improve frozen semen quality and fertility.

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski's horse (Equus ferus przewalskii).

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4°C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P<0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P>0.05) between the Przewalski's (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose-EDTA-glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4CN (CN=mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski's and 49.3%, 24.6%, 88.9% and 25.8CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P>0.05) among treatments across species, mitochondrial membrane potential was higher (P<0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski's stallion sperm expressed higher (P<0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski's stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski's and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski's horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.

Intratesticular transplantation of allogenic mesenchymal stem cells mitigates testicular destruction after induced heat stress in Miniature-horse stallions.

Testicular degeneration (TD) is the most frequent cause of sub or infertility in stallions. Currently, mesenchymal stem cells (MSC) have been studied as a therapeutic option for several diseases including induced-TD in laboratory animals. Therefore, this study aimed to evaluate the effect of intratesticular MSC therapy on the testicular histology of stallions submitted to scrotal heat stress. Ten healthy Miniature-horse stallions were submitted to testicular heat stress induced by a heating wrap device (42-45°C). Afterward, the stallions were divided into two groups and treated seven days later. MSCs-treated stallions were treated with an intratesticular injection of 10 × 106 of MSCs diluted in 5 mL of PBS, whereas placebo-treated stallions had 5 mL of PBS intratesticular injected. All stallions had testicular biopsies collected seven days before and one- and 14-days post-heat stress and were castrated 30 days after testicular insult. Tissue sections were stained with H&E and evaluated for the tubular and luminal diameter, epithelial thickness, seminiferous tubules (STs) integrity, the number of spermatozoa in the STs, and the percent of abnormal STs. Significance was set at P≤0.05. In both groups, testicular heat stress damaged the STs (P<0.05). However, STs' parameters were improved in MSCs-treated stallions compared to placebo-treated stallions 30 days after the testicular insult (P<0.05). In conclusion, the results of the present study suggest that intratesticular MSC therapy provided a therapeutic advantage in rescuing acute TD in stallions. However, further studies are essential to evaluate the benefits of this therapy on semen parameters and stallions with idiopathic TD.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen.

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions.

BACKGROUND: Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions. OBJECTIVE: To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders. STUDY DESIGN: Controlled and field study. METHODS: In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression. RESULTS: Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001). MAIN LIMITATIONS: Low number of mares in the fertility trial. CONCLUSION: Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.

Cross-cultural adaptation of the web-based executive functioning questionnaire for Brazilian sample (Webexec-BR).

The Web-based Executive Functioning Questionnaire (Webexec) is a brief scale developed to assess executive functions via online format. It has been used in different contexts, but its adaptation to other cultures is still restricted. This study aimed to perform a cross-cultural adaptation of the Webexec for a Brazilian sample considering the psychometric properties of the scale. This study used a sample of 295 Brazilian participants, with a mean age equal to 20.69 (SD = 6.030). This is a longitudinal study with reapplication of the scale six weeks after the test phase. Classical and contemporary methods were applied to analyze the psychometric properties of the Webexec. The results showed that the scale presented excellent psychometric properties for the Brazilian version, considering validity evidence based on the content and internal structure of Webexec, as well as reliability and precision. However, it is considered that other relational and experimental studies should be carried out with a larger sample size and for different population groups.

Impact of cryopreservation protocols (one- and two-step) on boar semen quality at 5 °C and post-thawing.

The two-step protocol (2 S) is currently used for boar semen cryopreservation. In this method, the cryoprotectant penetrant agents (CPAs) are added at 5 °C to reduce the toxicity of CPAs. An alternative is the one-step protocol (1 S), which is easier, cheaper, and reduces the necessity of equipment, but could increase the toxicity of CPAs. Currently, there are no studies that compared both protocols for boar semen cryopreservation. This experiment aimed to study the effect of cryopreservation protocol (1 S vs 2 S) on boar spermatozoa. In the one-step protocol, after centrifugation, the spermatozoa pellet was resuspended at 17 °C in the extender containing CPAs to achieve a concentration of 1 × 109 spermatozoa/mL and then submitted to cryopreservation. For the two-step protocol, the sperm pellet was resuspended in fraction A at 17 °C to achieve a concentration of 1.5 × 109 spermatozoa/ mL, and then allowed to cool to 5º C before fraction B with CPA was added to the sample to achieve a final concentration of 1 × 109 spermatozoa/mL and followed by freezing. The cryopreservation protocol did not impact total motility at 5 °C (1 S: 78.5 % vs 2 S: 79 %, p > 0.05). After thawing, the two-step protocol improved (p < 0.05) total (1 S: 18.2 % vs 2 S: 29.5 %) and progressive motility (1 S: 9 % vs 2 S: 15%). Further, the 2 S protocol increased (p < 0.05) the percentage of rapid spermatozoa (1 S: 8.7 % vs 2 S: 14.6 %) and spermatozoa with intact plasma and acrosomal membrane (IAIP) (1 S: 40.5 % vs 2 S: 61.5 %), and increased (p < 0.05) live sperm cells with high mitochondrial potential (MHIP) (1 S: 42.9 % vs 2 S: 60 %). The boar semen cryopreservation method (TRT) did not (p > 0.05) alter membrane lipid disorder, lipid peroxidation, and superoxide anion. Thus, the best method for boar semen cryopreservation is the two-step protocol.

Description and genetic analysis of three sets of monozygotic twins resulting from transfers of single embryos to recipient mares.

CASE DESCRIPTION: 3 sets of monozygotic twins resulting from transfers of single embryos to recipient mares were examined. CLINICAL FINDINGS: In all 3 recipient mares with twin pregnancies, only 1 embryonic vesicle was detected before day 25 of gestation. In 1 recipient mare, 2 apparent adjacent vesicles, each containing an embryo with a heartbeat, were visualized on ultrasonographic examination on day 37 of gestation. The other 2 recipient mares underwent ultrasonographic examination on day 30 of gestation, at which time only 1 vesicle and embryo was identified. In these latter 2 recipient mares, however, a thorough ultrasonographic examination for a second conceptus on day 30 had not been performed, as only 1 embryo had been transferred and visualized on early ultrasonographic examination. TREATMENT AND OUTCOME: All twin pregnancies resulted in death of both fetuses. Genetic analysis confirmed that each set of monozygotic twins originated from the transferred embryo. CLINICAL RELEVANCE: Monozygotic twin pregnancy may occur after embryo transfer; thus recipient mares should be examined thoroughly for multiple conceptuses, especially between 25 and 30 days of gestation. At this time, the allantoides of monozygotic twins should be visible ultrasonographically and effective management may still be possible.

The Combination of hCG and GnRH Analog to Hasten Ovulation in Mares Does not Change Luteal Function and Pregnancy Outcome in Embryo Recipient Mares.

Equine practitioners often prescribe the combined use of hCG and GnRH to hasten ovulation due to presumed synergistic effects. Therefore, this study aimed to test whether the combination of hCG and deslorelin acetate to hasten ovulation in mares would show any effect in inducing ovulation more efficiently than when either drug is used by itself, and to verify whether this association would affect progesterone concentrations; corpus luteum (CL) diameter and blood flow; and pregnancy outcome in recipient mares after embryo transfer (ET). Seventeen mares had the ovulation hastened (≥35 mm follicle) as follow: Control, 1 mL of 0.9% NaCl solution; GnRH, 1 mg of deslorelin acetate; hCG, 1,500 IU of hCG; hCG+GnRH, 1mg of deslorelin acetate and 1,500 IU of hCG. CL diameter and blood flow, and serum progesterone concentrations were assessed between the day of ovulation induction and sixteen days after ovulation. In addition, data of 194 ET were retrospectively analyzed. Pregnancy rates at five days after ET and pregnancy loss up to 60 days of recipient mares with natural ovulation (Control, n=37), or with ovulation hastened with hCG (n=25), or deslorelin acetate (n=46), or the combination of these hormones (n=86), as described above, were assessed. The control group had a higher progesterone concentration on the day of ovulation than the GnRH group (P < .05). However, there were no differences in CL diameter and blood flow at any time point, as well as in progesterone concentration over time (P > .05). Pregnancy rates and pregnancy loss didn't differ between recipient mares treated or not with hormones. In conclusion, the combination of hCG and deslorelin acetate to hasten ovulation was not able to change luteal development, progesterone concentration, or pregnancy outcome in recipient mares after ET.

Three Manual Noncommercial Methods to Prepare Equine Platelet-Rich Plasma.

In light of PRP's increasing popularity in veterinary practice, this study aimed to compare three manual methods to prepare and cool equine PRP. The blood of 18 clinically healthy mares was collected via venipuncture in a blood transfusion bag (method 1), blood tubes (method 2), and a syringe (method 3). In method 1, samples were double centrifuged; method 2 involved one centrifugation, and in method 3 the syringe was kept in an upright position to sediment for 4 h. After processing with three methods, PRP and platelet-poor plasma (PPP) were extracted and assessed for red (RBC) and white blood cell counts (WBC), platelet counts, and viability. In a subset of mares (n = 6), samples were processed with the three methods, and PRP was evaluated at 6 and 24 h postcooling at 5 °C. Method 1 resulted in the highest and method 3 in the lowest platelet concentration (p < 0.05), and the latter also had greater contamination with WBC than the others (p < 0.001). Platelet viability was similar across treatments (p > 0.05). Cooling for 24 h did not affect platelet counts in all methods (p > 0.05); however, platelet viability was reduced after cooling PRP produced by method 3 (p = 0.04), and agglutination increased over time in all methods (p < 0.001). The three methods increased (1.8-5.6-fold) platelet concentration in PRP compared to whole blood without compromising platelet viability. In conclusion, all three methods concentrated platelets and while cooling affected their viability. It remains unknown whether the different methods and cooling would affect PRP's clinical efficacy.

Allogenic mesenchymal stem cell-conditioned medium does not affect sperm parameters and mitigates early endometrial inflammatory responses in mares.

This study aimed to evaluate the effects of mesenchymal stem cell-conditioned medium (MSC-CM) on sperm parameters, intrauterine polymorphonuclear neutrophils (PMN), intrauterine fluid accumulation (IUF), and fertility in mares. In experiment 1, two ejaculates from ten stallions were extended to 50 million sperm/mL using a milk-based extender. Thereafter, 20 mL of extended semen was added of MSC-CM as follows: 0, 5, 10, 15, and 20 mL. Sperm kinetics and plasma membrane integrity were evaluated immediately after dilution (T0) and 2 h post-incubation at 37 °C (T2). In experiment 2, mares characterized as resistant (n = 13) or susceptible (n = 7) to endometritis were inseminated with fresh semen 24 h post-induction of ovulation in two (Control and CM-1) and three (Control, CM-1, and CM-2) cycles in a crossover, as follows: control, no pharmacological interference; CM-1, supplementation of semen insemination dose at 3:4 (v:v, MSC-CM:semen); CM-2, 30 mL of MSC-CM was infused into the uterus 24 h before insemination. Endometrial cytology and uterine fluid were collected 6 and 24 h after insemination to evaluate the number of PMNs and concentrations of interleukins IL6, IL10, and TNFα. IUF was determined by ultrasonography 24 and 48 h after insemination. Pregnancy status was diagnosed 14 days after ovulation. The addition of MSC-CM to semen did not influence sperm parameters at T0 and T2 (P > 0.05) and reduced (CM-1; P < 0.05) the number of PMNs at 6 h post-insemination in resistant mares. In susceptible mares, PMNs at 6 and 24 h post-insemination, as well as IUF were reduced (P < 0.05) in both treated cycles (CM-1 and CM-2). In addition, MSC-CM downregulated IL6 and upregulated IL10 concentrations in the uterus of susceptible mares after insemination. There were no differences in fertility rates among groups both in resistant (Control, 77%, 10/13; CM-1, 62%, 8/13) and susceptible mares (Control, 42.8%, 3/7; CM-1, 57.1%, 4/7; CM-2, 85.7%. 6/7). In conclusion, MSC-CM did not affect sperm parameters when mixed with diluted semen, and reduced post-insemination inflammatory responses in mares.

Use of Intravaginal Progesterone-Releasing Device Results in Similar Pregnancy Rates and Losses to Long-Acting Progesterone to Synchronize Acyclic Embryo Recipient Mares.

The objectives of this study were: (1) to assess uterine features and serum progesterone concentrations of acyclic mares synchronized and resynchronized with intravaginal progesterone release device (IPRD), and (2) to compare pregnancy rates and losses of cyclic and acyclic embryo recipient mares treated with different synchronization protocols. In Experiment 1, mares (n = 12) received estradiol for 3 days (E2-3d), and then 24 h after the last injection, an IPRD was inserted and kept in place for 9 days. Three days after IPRD removal, mares were treated with E2-3d, and then a new IPRD was inserted and maintained for three days. Serum progesterone concentrations were assessed 2, 6, and 12 h after insertion and removal of IPRD, and then daily from the insertion of the first IPRD to one day after removal of the second IPRD. Experiment 2 was conducted with embryo recipient mares randomly assigned to four groups: (1) Cyclic: mares (n = 75) had ovulation confirmed after receiving a single dose of histrelin when a periovulatory follicle was first detected, (2) LAP4: acyclic mares (n = 92) were treated with E2-3d and then administered a single dose of LAP4 24 h after the last estradiol injection, (3) IPRD: acyclic mares (n = 130) were treated with E2-3d and an IPRD for 4-8 days, and (4) RE-IPRD: acyclic mares (n = 32) were synchronized as in the IPRD group but not used for embryo transfer (ET), then 8 to 15 days later, the mares were resynchronized with E2-3d and an IPRD for 4-8 days. In vivo-produced Day-8 embryos were collected and transferred 4-8 days after ovulation or progesterone treatments. Mares in IPRD and RE-IPRD groups had the intravaginal device removed immediately before ET, and then a new IPRD was inserted right after ET. Pregnancy diagnosis was performed at 5, 30, and 60 days after ET. Once pregnancy was confirmed, mares in the three acyclic groups received weekly injections of LAP4 (1.5 g) until 120 days of pregnancy. Mares in IPRD and RE-IPRD groups had the device removed three days after the first pregnancy diagnosis. In Experiment 1, progesterone concentrations increased rapidly starting 2 h after insertion of IPRD (p < 0.05); then, concentrations plateaued well above pregnancy maintenance until removal on days 9 and 3, respectively. Progesterone concentrations were reduced to baseline 24 h after IPRD removal (p < 0.05). For experiment 2, there was no difference in pregnancy rates across groups (65-74%) or pregnancy losses by 60 days of gestation (7-12%) (p > 0.05). In conclusion, the IPRD used herein resulted in a rapid increase and a sharp decline in progesterone concentrations upon its insertion and removal, respectively. Finally, our results demonstrated that IPRD could be a compatible alternative to LAP4 to synchronize and resynchronize acyclic embryo recipient mares.

Primary Uterine Hemangiosarcoma in a Thoroughbred: A Case Report.

A 1.8-year-old maiden Thoroughbred filly, without previous history of mating or reproductive management, was referred for clinical inspection due to the presence of sanguineous vaginal discharge and severe abdominal pain. Transrectal palpation indicated uterine asymmetry, and transrectal ultrasonography revealed a mass near the cervix measuring 8.3 cm in diameter, with heterogeneous echogenicity, a trabeculated center, and a well-defined hyperechoic border. Smaller masses surrounded the larger uterine mass. During the examination, the mare expelled a uterine mass through the vulva. Histological and immunohistochemical (CD31 and Factor VIII) examinations of the expelled mass suggested a diagnosis of hemangiosarcoma. Therefore, a therapeutic hysterectomy was performed, and examinations of the uterine tissue confirmed the diagnosis. However, the mare was euthanized 2 weeks later due to postoperative complications. The animal was subjected to necropsy, and intestinal adhesions in the surgical incision were diagnosed as postoperative complications. No other neoplasms were found during necropsy, establishing the primary origin of the tumor. This case study presents the first known report of uterine hemangiosarcoma in an equine species.

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares.

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (-48 and -24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilß, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey's post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property.

Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares.

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility.

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk-based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one "bad cooler" and one "good cooler" stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.

Update on Seminal Vesiculitis in Stallions.

Seminal vesiculitis in stallions reduces fertility and is often underdiagnosed. The most common cause is infection of seminal vesicles by bacteria capable of forming biofilms and a propensity for tissue persistence, for example, Pseudomonas aeruginosa. Achieving a clinical cure is challenging because of a high rate of recurrence. Systemic antibiotic therapy does not reach adequate therapeutic concentrations within the seminal vesicles; one alternative is endoscopy-guided, local antibiotic infusion into the gland lumen, with or without concurrent systemic antibiotics. Current diagnostic and therapeutic strategies for seminal vesiculitis are less than fully satisfactory, and several studies have been conducted to improve them. This review covers traditional and newer concepts regarding seminal vesiculitis, including diagnostic and treatment methods, management of stallions with this disorder, and authors' experience with clinical cases.

Fractionated semen collection as a tool to rescue fertility in stallions with seminal vesiculitis.

Treatments for seminal vesiculitis have poor outcomes in stallions; thus, the development of alternative strategies is warranted. This study aimed to evaluate fractionated semen collection as a method to restore the fertility of stallions diagnosed with seminal vesiculitis. Eighteen ejaculates from six stallions (three ejaculates/stallion) diagnosed with seminal vesiculitis were harvested in fractions, as follows: Fraction A (FA), the first two jets; Fraction B (FB), the third and fourth jets; and Fraction C (FC), the fifth and remaining jets of the ejaculate. All fractions were subject to standard semen evaluations that were performed in addition to cytology and bacterial aerobic cultures. Fractions were extended and cooled to 5 °C. As a proof of concept, 20 mares (48 estrous cycles, ∼8 cycles/stallion) were bred with 1 billion sperm from FA (cooled at 5 °C for 24 h). In our study, FA had negative bacterial cultures, absent macroscopic or microscopic abnormalities; FB had positive bacterial cultures in two stallions and presence of polymorphonuclear neutrophils (PMNs) in all samples, but with no macroscopic abnormalities; and FC had positive bacterial cultures, purulent appearance, and the presence of degenerated PMNs, just as noted in the whole semen. Overall, post-cooling sperm motility results were superior (P < 0.05) for FA in comparison with FB and FC. First cycle pregnancy rates using FA varied from 66% to 86%. None of the non-pregnant mares developed endometritis. In conclusion, fractionated semen collection can be used to obtain semen free of contamination and to achieve satisfactory pregnancy rates from stallions with seminal vesiculitis.