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 (Ilβ, 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

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

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

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

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 p > 0.05). This study helps to clarify and define normal estrous characteristics of jennies in the Eastern Caribbean

Data_Sheet_1_Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.docx

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 (O2−) 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.</p