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.

Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.

Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide ( O 2 - ) were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P < 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P < 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P < 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.

Evaluation of Stress Accompanying Immunocontraceptive Vaccination in Donkeys.

The overpopulation of donkeys is recognized as a problem in many parts of the world. The main concerns with uncontrolled donkey populations are habitat degradation and competition for feed resources between donkeys and other species. One of the most effective and humane solutions is the use of immunocontraception. Therefore, this study sought to evaluate the stress imposed by the use of two formulations of a zona pellucida (ZP) vaccine, a recombinant (reZP) and a native porcine (pZP) vaccine, both formulated with a Freund's adjuvant. The stress was objectively measured using fecal cortisol concentrations and physical examination parameters at fixed points before and after vaccination. We hypothesized that fewer changes in physical exam parameters and lower fecal cortisol concentrations would be stimulated in jennies treated with the reZP vaccine due to the selection of specific proteins. Twenty-five reproductively sound jennies were randomly assigned to reZP (n = 9), pZP (n = 8) or control (n = 8) groups. The vaccines were administered at five-week intervals. Physical exam parameters and body wall thickness of injection sites were recorded for each jenny for four days post-injections. Fecal samples were obtained every other day from day 0 (first vaccination) through day 6 and on days 35 to 41 after booster. Injection site reactions were common in all groups with the reZP and pZP groups being overrepresented. Lameness was observed in the pZP and reZP groups that were affected by injection site reactions and open abscesses. The present study showed an increase in fecal cortisol concentrations within 4 days after the first vaccination with ZP vaccines and, thereafter, a decrease in cortisol 35 days later after the second vaccination, especially in donkeys with open abscesses. Our results suggest that acute stress (increased cortisol) was induced after the first vaccination, and chronic stress (decreased cortisol) occurred thereafter in association with open abscesses. In conclusion, reZP and pZP formulated with Freund's adjuvant induced local inflammatory reactions with a differential degree of acute and chronic stress in donkeys.

Early Pregnancy in Jennies in the Caribbean: Corpus Luteum Development and Progesterone Production, Uterine and Embryo Dynamics, Conceptus Growth and Maturation.

We aimed to characterize early embryo development and changes in corpus luteum (CL) development and progesterone profile in pregnant vs. non-pregnant jennies. Eight jennies were enrolled in the study. In the first two cycles, the jennies were monitored by transrectal ultrasonography and had blood harvested for hormone profile assay. In the third cycle, jennies were bred by a jack of proven fertility. Jennies were then monitored and sampled for up to 30 days of pregnancy. Data were evaluated by random-effects multiple linear regression, and correlations were expressed as Pearson's correlation coefficient. Progesterone concentration rose rapidly from ovulation (D0) until D7, plateaued until D12-14, then precipitously declined between D14 and 15, remaining low until the next ovulation in non-pregnant cycles. In the pregnant jennies, the progesterone concentration rose to maximal concentrations on D7-11, being higher at this stage than in non-pregnant cycles, then declined gradually up to D30. In all cycles, the volume of the CL increased steadily until D6, when it plateaued in pregnant jennies. For non-pregnant jennies, CL volume decreased slowly from D6 to D11 and then had a faster drop. Uterine tone increased following ovulation, becoming turgid around the day of embryo fixation (D15.0 ± 0.9). An embryonic vesicle (EV) was first detected on D9.3 ± 0.5 (2.4 ± 0.5 mm). The EV remained spherical until D18.6 ± 1.4. The embryo proper was first detected ventrally in the vesicle on D20.8 ± 1.1 and the embryonic heartbeat by D22.0 ± 0.9. The allantoic sac was identified at D24.0 ± 0.9, and at D30, the allantoic sac filled the ventral half of the EV. This study provides evidence that higher cumulative concentrations of progesterone are correlated to size of the EV, and there were changes in the luteal dynamics and progesterone profiles in pregnant vs. non-pregnant jennies.

In Vitro Antimicrobial Activity of Selected Essential Oils Against Endometritis-Causing Microorganisms in Mares.

This study aimed to evaluate the in vitro antimicrobial activity of essential oils (EO) from Ocimum basilicum (basil), Rosmarinus officinalis (rosemary), and Cymbopogon citratus (lemongrass) on endometritis-causing microorganisms in mares. Serial concentrations of the EO from 30.00 mg/mL to 0.47 mg/mL were tested. The major compounds of O. basilicum EO were linalyl acetate (33.32 wt.%) and citronellal (25.06 wt.%); of R. officinalis EO were borneol (26.48 wt.%), trans-ß-ocimene (16.76 wt.%), camphene (12.45 wt.%), and α-phellandrene (11.08 wt.%); and of C. citratus EO were geranial (45.96 wt.%) and neral (32.62 wt.%). Regarding antimicrobial activity, C. citratus EO has had the highest inhibition percentage (73.9%), followed by O. basilicum (67.2%) and R. officinalis (58.7%). P. aeruginosa was the only pathogen unable to establish the minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) values for the studied EO. The EOs were effective against all other microorganisms (S. equi, S. aureus, K. pneumoniae, E. coli, and C. Albicans). In conclusion, the EOs of O. basilicum, R. officinalis, and C. citratus have presented in vitro antimicrobial activity against microorganisms causing endometritis in mares.

Structural and Functional Dynamics of the Ovary and Uterus during the Estrous Cycle in Donkeys in the Eastern Caribbean.

Eight non-bred, non-pregnant, regularly cycling Caribbean jennies were examined daily via transrectal ultrasound to define the ovarian and uterine dynamics during four consecutive estrous cycles. Blood samples were collected every other day for progesterone analysis. The mean (±SD) overall inter-ovulatory interval across all donkeys and cycles was 22.93 ± 1.99 days. The maximum follicular diameter was 34.6 ± 2.9 mm. A two-wave pattern was evident in 97% (30/31) of the cycles. The emergence of the future dominant follicle and the largest subordinate follicle of the major primary wave coincided on Day 5.7 ± 3.6 post-ovulation, whereas the secondary wave emerged on Day 19.8 ± 2.9 during estrus of the previous cycle or early diestrus. The secondary wave was often minor (93%, 28/30 cycles). Follicular deviation occurred 8.2 ± 1.4 days before the subsequent ovulation. Luteal volume increased for the first four days after ovulation and reached a maximum volume of 8.5 ± 2.7 mm3 at Day 5.4 ± 0.4, before gradually regressing after Day 15. Serum progesterone concentration increased from Day 1 after ovulation, peaking at 27.0 ± 9.6 ng/mL between 7 and 10 days after ovulation. Progesterone concentration dropped precipitously around Day 15 after ovulation and was below 2 ng/mL around Day 17 ± 2. A day effect (p < 0.0001) was observed for corpus luteum's volume, progesterone concentration, and uterine tone, but not for endometrial edema (p > 0.05). This study helps to clarify and define normal estrous characteristics of jennies in the Eastern Caribbean.

Efficacy and Side Effects of Low Single Doses of Cloprostenol Sodium or Dinoprost Tromethamine to Induce Luteolysis in Donkeys.

Due to the limited literature available evaluating doses of Prostaglandin F2α in donkeys, doses for horses have been extrapolated and used as guidelines. This study aimed to assess the efficacy and side effects of four different cloprostenol sodium and dinoprost tromethamine doses to induce luteolysis in jennies. Sixty-three cycles of seven Jennies (nine cycles per jenny) were used in this study. Seven days after ovulation, jennies randomly received one of the treatments in a crossover design as follows: Control, no treatment was administered; C1, 250 µg of cloprostenol sodium (CS, Estrumate , Merck Animal Health, USA); C2, 125 µg of CS; C3, 65.5 µg of CS, C4, 37.5 µg of CS; DT1, 5 mg of dinoprost tromethamine (DT, Lutalyse, Zoetis, USA); DT2, 2.5 mg of DT; DT3, 1.25 mg of DT; DT4, 0.625 mg of DT. Jennies were monitored for 30 minutes following treatment, and adverse effects were recorded. The measurement of the corpus luteum (CL) and the length of the estrous cycle were recorded. All DT and CS treatment doses were effective (P < .0001) in reducing the estrous cycle length compared to jenny's Control cycle. The CL volume was decreased in all treated groups one day after treatment (P < .05). The adverse effects were reduced as the dose of both Prostaglandin F2α analogs were reduced. In conclusion, a single low dose of dinoprost tromethamine (0.625 mg) or cloprostenol sodium (37.5 µg) can induce luteolysis and shorten the estrous length in jennies producing fewer adverse effects.

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.

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.

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.

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.

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.

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions.

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck's length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.

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.

Alternative Protocol Using Imipramine, Detomidine, and Oxytocin for Semen Collection in Stallion with Ejaculatory Dysfunction.

A 7-year-old Quarter Horse stallion was admitted at the hospital with a history of ejaculatory failure for 12 months. The stallion revealed no physical or psychological abnormalities, as well as, normal libido and erection. In addition, there were no abnormalities in accessory sex glands or the aorta artery detected by transrectal ultrasonography. Based on clinical findings, the stallion was diagnosed with an idiopathic ejaculatory dysfunction; therefore, alternative attempts of semen collection were performed. Thermal compress on the basis of the stallion's penis, semen collection on the ground, and imipramine hydrochloride treatment were performed unsuccessfully. However, a protocol consisted by the association of imipramine (3 mg/kg/v.o.), detomidine (0.02 mg/kg/i.v.), and oxytocin (20 U.I./i.v.) successfully produced ejaculation in this stallion. The semen obtained from ex copula ejaculation of the stallion was collected using a collector cup lined with a plastic bag, which was positioned over the prepuce of the stallion. Semen with good sperm quality (87% of total motility) was obtained using the proposed protocol. Semen was then processed for cryopreservation and post-thawed semen samples presented satisfactory sperm parameters. In conclusion, the association of imipramine, detomidine, and oxytocin can be considered for ex copula semen collection in stallions.

Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender.

The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour-cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.

Comparative Efficacy of Histrelin Acetate and hCG for Inducing Ovulation in Brazilian Northeastern Jennies (Equus africanus asinus).

The goal of this study was to compare the efficiency of histrelin acetate (GnRH analog) and human chorionic gonadotropin (hCG) to hasten ovulation in Brazilian Northeastern jennies (Equus africanus asinus). Thirty cycles of ten jennies were randomly assigned in one of the three groups: G0 (control group), saline; G1, 250 µg of histrelin acetate; G2, 2500 IU of hCG. Jennies were evaluated by transrectal palpation and ultrasonography, and had the administration of an ovulation-inducing agent when a follicle measuring between 29 and 32 mm of diameter was diagnosed. Jennies were monitored every 6 hours by transrectal ultrasonography until ovulation. The interval between prostaglandin administration and ovulation was lower (P < .05) in jennies from the G1 (145.2 ± 34.6 hours) and G2 (147.4 ± 27.3 hours) groups compared with the control cycle (220.0 ± 41.8 hours). Both treatments (G1, 41.15 ± 3.5 hours; G2, 37.8 ± 2.5 hours) also reduced (P < .05) the interval that jennies took to ovulate after the administration of the ovulation-inducing agent compared with the control (81.8 ± 28.8 hours). All jennies from G1 and G2 ovulated up to 48 hours after ovulation induction, whereas 100% of jennies in the control cycle ovulated later (>48 hours from the administration of saline). In conclusion, both histrelin acetate and hCG at the used dose are efficient ovulation-inducing agents in jennies promoting ovulation up to 48 hours after administration.

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.

An approach to rescue the fertility of stallions with a high level of hemospermia.

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control-pure raw semen, (b) WB50-50% (v/v) whole blood added into semen, (c) E1-WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2-WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O2 ) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p < .05) in E1 than in CT and E2 samples. Progressive motility decreased (p < .05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p > .05); however, O2 production was higher (p < .05) in WB50 than in E2 samples, while the values were intermediate (p > .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p < .05) fertility than the others (WB50-0% and E1-25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.