Recipient Mares Research Articles

104 Induction of cyclicity in seasonally anestrous recipient mares

Anestrous (AN) recipient mares’ natural cyclicity must be manipulated to produce foals from transferred embryos early in the year. One approach uses sulpiride to stimulate prolactin (PRL) that appears to have an important reproductive role effecting seasonality in mares. While previous research utilized sulpiride to induce ovulation (OV) in AN mares, few have transferred embryos after treatment. This study was designed to evaluate the effect of a treatment of estradiol cypionate (ECP) and sulpiride on inducing OV and subsequent pregnancy outcome in AN recipient mares of various reproductive status performed on a commercial embryo transfer farm. Anestrous recipient mares (n = 32) were randomly assigned to 4 treatment groups based on records of ovarian activity. Transitional (T, n = 8), AN (n = 11), and progesterone pre-treated (P, n = 9, received 4 mg altrenogest IM 3 wk prior) groups were treated January 20 with 50 mg of ECP IM and 3 g of sulpiride IM 24 h later. A control group (C, n = 4) of AN mares received the carrier. Jugular blood samples were collected at 0, 3, 6, 12, and 24 h post-treatment followed by daily collection for 10 d and collection every other day for 32 d thereafter and analyzed for luteinizing hormone (LH) and PRL. Ovaries were transrectally scanned every 2 to 3 d for 60 d. Embryos were transferred to 10 mares (5 T, 3 P, 2 AN) after at least the second estrous cycle up to the sixth cycle depending on their synchrony to the donor. Binomial data was analyzed via PROC GLIMMIX of SAS, and number of days to OV and hormonal data were analyzed via PROC MIXED with repeated measures. Nonparametric data were natural log-transformed and analyzed if normality was improved. Differences of least squares means were determined using the PDIFF option in SAS with ∝ ≤ 0.05 used to declare significance. There was an effect of treatment on the number of mares that achieved first OV ( P < 0.001) with 23 of 28 (82.1%) treated mares having a first OV within 22 d of treatment; and second OV ( P = 0.018) with 15 of the 23 (65.2%) mares that had a first OV achieving a second OV within 43 d. The mean number of days to first OV of T, AN, and P groups together were 32 ± 5 d earlier ( P < 0.001) than C. The transitional group ovulated sooner ( P = 0.05) than AN and tended ( P = 0.07) to ovulate sooner than P. There was an effect of the treatment-by-day interaction on plasma mean PRL ( P < 0.001) and LH ( P = 0.021) concentrations. Of the 10 embryos that were transferred, 7 (70%) resulted in a confirmed pregnancy at 60 d. Overall, the treatment protocol using ECP and sulpiride appeared to hasten OV and resulted in sustained pregnancy after embryo transfer.

Correlations of corpus luteum blood flow with fertility and progesterone in embryo recipient mares.

The aim of this study was to evaluate the correlation between the corpus luteum vascularization with the concentration of progesterone and the fertility of embryo recipient mares. Mangalarga Marchador mares (n = 33) were distributed into groups according to the days (D) after ovulation, as follows: D3 (n = 8), D4 (n = 8), D5 (n = 9), and D6 (n = 8). The evaluations of the corpus luteum, endometrium, and blood collection to quantify the progesterone concentration were carried out on D3, D4, D5, and D6. Among the parameters evaluated, only progesterone concentration on D6 differed from the other groups (P <0.05). A positive correlation (P <0.05) between the diameter and the area of the corpus luteum, and the objective and subjective methods of the corpus luteum vascular perfusion, was identified. Likewise, a positive correlation (P <0.05) was observed between the objective and subjective methods of the vascular perfusion in the corpus luteum and the progesterone concentration. The pregnancy rate obtained in this study (54.54%) was not affected (P> 0.05) by the day of embryo transfer, whose percentages were 37.50% (3/8) on D3, 50% (4/8) on D4, 66.70% (6/9) on D5, and 62.50% (5/8) on D6. It was estimated that with each increase on the day of embryo transfer, the pregnancy rate increases. The results allow to conclude that the corpus luteum vascularization in mares, evaluated by Doppler ultrasound, correlates with progesterone concentration and the embryo transfer day.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Simple SummaryObesity has been associated with altered reproductive activity in mares and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

Successful vitrification of manually punctured equine embryos.

Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. To develop a manual method of blastocoele collapse prior to vitrification using commercial products. In vivo experiment. Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non-punctured≤300µm (n=10) and>300 to≤560µm (n=10), punctured small (>300 to≤560µm; n=17) and large (>560µm; n=10) and exposed to the equilibration solution (ES) in the kit for 6min. An additional group of punctured large embryos was exposed to ES for 8min (n=10). For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature. Vitrified, manually punctured embryos≤560µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos≤300µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos>560µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Limited group sizes. High pregnancy rates can be achieved by manually puncturing≤560µm equine embryos prior to their vitrification and subsequent warming in commercial media.

Evaluation of Corpus Luteum Vascularization in Recipient Mares by Using Color Doppler Ultrasound

Background: Embryo transfer is one of the most commonly used reproductive biotechnique. The success of embryo transfer is also affected by the synchrony of estrus and ovulation between donor and recipient animals. In horse reproduction, ultrasonography has been used, among other purposes, to diagnose early pregnancy. However, only the color Doppler imaging mode makes it possible to evaluate the vascular architecture and the hemodynamic aspects of the vessels in several organs, especially the corpus luteum. The objective of this study was to evaluate, based on the color Doppler ultrasound, the corpus luteum vascularization and function from recipient mares at embryo transfer timing.Materials, Methods &amp; Results: Mangalarga Machador mares from 5 to 10-year-old and a range of live weights of between 350 to 450 kg were used for this experiment, kept in pasture-based on mombaça grass (Panicum maximum) and were given ad libitum access to water and mineral supplementation. The animals (n = 15) were gynecologically examined and uterine consistency was evaluated by rectal palpation the same operator using an ultrasound system (SonoScape®) with a linear transducer, and operating frequency ranging from 5 to 10 Mhz. The uterine tone was classified between grades 1 and 4 and subjected to ovulation induction. The objective and subjective vascular perfusion of the corpus luteum was evaluated by color Doppler ultrasound on the day of embryo transfer and endometrium. The determination progesterone concentration on the day of the embryo transfer was performed by direct chemiluminescence assay. The arcsine (√P/100) transformation was applied to the percentage data, and the results were expressed as mean (.) ± standard error of the mean (SEM). Further, the assumptions of normality and homoscedasticity were verified, respectively, based on the Shapiro-Wilk and Lilliefors tests. Regarding the parametric and non-parametric variables, were applied, respectively, analysis of variance (ANOVA) followed by Tukey’s test, and the Kruskal-Wallis test followed by Dunn’s test. Pearson’s correlation coefficient was used to evaluate the relationship between the parameters. The statistical program SPSS 16.0 was used to perform the over-mentioned analyses, and a p-value &lt; 0.05 was taken as significant. Corpus luteum vascular perfusion, based on the objective and subjective evaluation methods, and the progesterone concentration were higher in the pregnant mares (P &lt; 0.05). The objective and subjective methods for evaluation of the vascular perfusion in the corpus luteum were positively correlated between themselves as well as to progesterone concentration (P &lt; 0.05). There was no significant difference between the groups considering the uterine tonus evaluation (P &gt; 0.05).Discussion: Mares that later became pregnant showed a higher concentration of progesterone as an outcome of the higher vascularization in the corpus luteum. It can be supported by both the correlation between the progesterone concentration and the corpus luteum vascular perfusion, as well as by the higher values of the vascular perfusion in pregnant mares. Based on the results, it has been concluded that the color Doppler ultrasound evaluation is an accurate tool to determine the corpus luteum vascularization, whether considering the objective or subjective methods. Also, the vascular perfusion is the most efficient parameter to determine both the corpus luteum function and to predict the ability of the recipient mares to maintain pregnancy.

Serum Progesterone and Conception Rates in Acyclic Embryo Recipient Mares Using a Bovine Progesterone-Releasing Intravaginal Device

The objective of this study was to quantify serum progesterone levels, uterine features, and pregnancy rates in acyclic, embryo recipient mares using a bovine progesterone-releasing intravaginal device in a commercial embryo transfer (ET) program. The study included 73 recipient mares of unknown breed, aged 3–10 years, weighing 350–500 kg, and kept under an intensive management system on Tifton 85 (Cynodon spp.) pastures with water and mineral salt ad libitum. The horses were divided into two groups: a group with a progesterone-releasing intravaginal device (1 g progesterone, G-IVP4, n = 24) and a control group (G-iP4, n = 49) receiving an injection of 1,500 mg long-acting progesterone. Jugular blood was collected for the G-IVP4 group for subsequent progesterone measurement by radioimmunoassay on three occasions: Day 0 (D0), intravaginal device was placed; Day 5 (D5), day of the ET; and Day 9 (D9), day of pregnancy diagnosis. There was an increase (P < .0001) in serum progesterone levels on D5 and D9 compared with D0 (4.09 ± 0.81 and 6.45 ± 1.03 ng/mL vs. 0.71 ± 0.14 ng/mL). There were no differences among groups in the pregnancy rate (P > .05), with rates of 83.33% and 73.46% for G-IVP4 and G-iP4, respectively. In conclusion, the intravaginal route for absorption of 1 g of progesterone device increased the serum level of progesterone sufficiently to prepare the uterus of acyclic recipient mares for ET, and the conception rate was similar to the standard protocol using long-acting injectable progesterone.

Relationships between blood and follicular fluid urea nitrogen concentrations and between blood urea nitrogen and embryo survival in mares

High blood urea nitrogen (BUN) concentration is linked to low fertility in cows and ewes; however, this relationship has not been reported in mares. The study characterized the relationship between BUN and follicular fluid urea nitrogen (FUN) during follicle growth (Experiment 1) and the impact of BUN from embryo donors on the pregnancy outcome of recipient mares (Experiment 2). In experiment one, follicular fluid and blood samples were collected from mares during diestrus with growing follicles and during estrus with pre-ovulatory follicles (n = 16 and 10 mares, respectively). In experiment two, BUN concentrations of embryo donors were related to pregnancy outcome after embryo transfer. In experiment one, there was a strong positive correlation between BUN and FUN (R = 0.83; P < 0.0001), with higher BUN in mares with growing follicles than with preovulatory follicles (P = 0.004) and higher FUN in growing follicles than in preovulatory follicles (P = 0.031). In experiment two, BUN was higher in donor mares that produced unsuccessful embryos compared to donor mares that produced embryos resulting in successful pregnancies at D14 (P < 0.03). Additionally, there was an effect of age (P = 0.01) and interaction between age and lactation (P = 0.009) in donor mares for embryo survival after embryo transfer. Donor mares with unsuccessful embryos were older than donor mares with successful embryos. Therefore, these experiments showed that BUN was related to follicular fluid environment as well as to the survival of Day 7–8 embryos after transfer to recipient mares.

Ultrasound-guided aspiration of dominant follicles (≥25 mm) followed by luteinization and progesterone production during the estrous cycle in mares

The present study was designed to evaluate luteinization rates subsequent to aspiration of dominant follicles (≥25 mm) in the absence of a functional CL (progesterone <1 ng/mL) and characterize the temporal changes in plasma concentrations of progesterone following aspiration-induced luteinization during the estrous cycle in mares. A total of 29 estrous cycles involving 15 mares in a cross-over design were randomly assigned to five groups: 1) ASP-F≥25 mm (n = 6; follicle aspiration 25–29 mm), 2) ASP-F≥30 mm (n = 6; follicle aspiration 30–34 mm), 3) ASP-F≥35 mm (n = 6; follicle aspiration 35–40 mm), 4) ASP-F≥40 (n = 6; follicle aspiration ≥40), and 5) Control (n = 5; spontaneous ovulation or no follicle aspiration). Subsequent to ovulation (Day 0), PGF was administered to all groups on Day 5, blood samples were collected daily and aspiration of the dominant follicle was done using ultrasound-guided transvaginal follicle needle puncture. Among the follicle aspirations groups 25–29, 30–34, 35–39, and ≥40 mm, the luteinization rates were not different (P > 0.05) at 83, 67, 83, and 100%, respectively. Correspondingly, progesterone concentrations increased (>2 ng/mL) by approximately 6, 7, 5, and 4 d after aspiration, respectively, which were delayed (P < 0.05) in the 25–29 and 30–34 mm follicle aspiration groups compared to 2 d after ovulation in the control group. Thereafter, progesterone reached maximal concentrations (10–11 ng/mL) as averaged over all aspiration groups but were lower (P < 0.05) compared to the mean maximal concentration (18 ng/mL) in the control group. Subsequently, there was a decrease in progesterone concentrations (<2 ng/mL) in response to luteolysis, which was delayed (P < 0.05) in the aspiration groups over Days 16–20 compared to Day 15 in the control group. Despite this discrepancy, the mean length of the interovulatory intervals were not different (P > 0.05) among groups on Day 23. Thus, the present study provided novel information that the luteinization rate is relatively high (83%) and consistent following aspiration of dominant follicles (≥25 mm) in the absence of a functional CL and that the increase in progesterone reaches sustainable progestational concentrations (≥2 ng/mL) in accord with the length of the estrous cycle that may potentially support development and maintenance of early pregnancy in recipient mares involved in an embryo transfer program.

Puncture of the Equine Embryonic Capsule and Its Repair In Vivo and In Vitro

Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured in vitro for 48 hrs (n = 3 per puncture group) or transferred to recipient mares and recovered 48 hrs later (n = 3 per puncture group). No pregnancies resulted from the 10 embryos punctured with the acupuncture needle, whereas 15 of 20 (75%) and 10 of 15 (67%) punctured on the micromanipulator or manually with the microneedle resulted pregnancies. Neither acupunctured nor microneedle-punctured embryos repaired their capsules in vitro. The acupunctured embryos also failed to repair their capsule after 48 hrs in vivo and subsequent uterine flushing yielded numerous capsular vesicles. The microneedle-punctured embryos did repair their capsule in vivo. Puncture with the microneedle opens the way for development of a manual method to vitrify equine embryos.

Effect of hCG administration in recipient mares at the day of embryo transfer aiming to increase pregnancy rates

Effect of hCG administration in recipient mares at the day of embryo transfer aiming to increase pregnancy rates

Evaluating the effect of ovine placental extract on early embryonic development and incidence of early pregnancy loss after embryo transfer in recipient mares

Advanced Glycation End-products (AGEs) are elevated in the uterine environment of obese infertile women. Can their detrimental effects on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)?Human endometrial epithelial cells (ECC-1) were exposed to AGEs at concentrations physiologically representative of the lean and obese uterine fluid, and three potential therapeutics: 25 nM RAGE antagonist FPS-ZM1, 100 μM metformin, and a combination of antioxidants (10 μM N-acetyl-L-cysteine, 10 μM N-acetyl-L-carnitine, and 5 μM α-lipoic acid). Real time cell analysis (xCelligence) determined the rate of adhesion and proliferation. Proliferation of organoid derived cells, and secretion of cytokines from organoids was characterized in the presence of AGEs (n=5). Uterine fluid of women undergoing assisted reproduction was profiled for AGEs-associated inflammatory markers (n ≥ 67 for each correlation).ECC-1 proliferation was reduced by obese versus lean AGEs and vehicle control (P = 0.04), and restored to lean conditions by antioxidants. AGEs influenced organoid derived primary endometrial epithelial cell proliferation in a donor dependent manner. AGEs increased organoid secretion of proinflammatory cytokines, including CXCL16 (P < 0.01). Clinically, CXCL16 correlated positively to maternal BMI (R = 0.26, P = 0.02) and intrauterine glucose (R = 0.74, P < 0.0001).Physiologically relevant concentrations of AGEs alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGEs-treated endometrial epithelial cells (ECC-1). Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and cytokine secretion in the presence of AGEs equimolar with the obese uterine fluid.

Likelihood of pregnancy is reduced in lactating recipient mares with a short post-partum period preceding embryo transfer

Likelihood of pregnancy is reduced in lactating recipient mares with a short post-partum period preceding embryo transfer

Effect of altrenogest supplementation on endogenous progesterone production in embryo recipient mares in a commercial embryo transfer program

To determine the physiologic and behavioral effects and pharmacokinetic profile of hydromorphone administered intravenously (IV) to horses.Prospective, randomized, crossover study.A group of six adult healthy horses weighing 585.2 ± 58.7 kg.Each horse was administered IV hydromorphone (0.025 mg kg–1; treatment H0.025), hydromorphone (0.05 mg kg–1; treatment H0.05) or 0.9% saline in random order with a 7 day washout period. For each treatment, physiologic, hematologic, abdominal borborygmi scores and behavioral data were recorded over 5 hours and fecal output was totaled over 24 hours. Data were analyzed using repeated measures anova with significance at p < 0.05. Blood samples were collected in treatment H0.05 for quantification of plasma hydromorphone and hydromorphone-3-glucuronide and subsequent pharmacokinetic parameter calculation.Hydromorphone administration resulted in a dose-dependent increase in heart rate (HR) and systolic arterial pressure (SAP). HR and SAP were 59 ± 17 beats minute–1 and 230 ± 27 mmHg, respectively, in treatment H0.05 at 5 minutes after administration. No clinically relevant changes in respiratory rate, arterial gases or temperature were observed. The borborygmi scores in both hydromorphone treatments were lower than baseline values for 2 hours. Fecal output did not differ among treatments and no evidence of abdominal discomfort was observed. Recorded behaviors did not differ among treatments. For hydromorphone, mean ± standard deviation for volume of distribution at steady state, total systemic clearance and area under the curve until the last measured concentration were 1.00 ± 0.29 L kg–1, 106 ± 21 mL minute–1 kg–1 and 8.0 ± 1.5 ng hour mL–1, respectively.Hydromorphone administered IV to healthy horses increased HR and SAP, decreased abdominal borborygmi and did not affect fecal output.

Estimation of the age of the early CL by ultrasonography improves the management of recipient mares in a commercial ET program

Estimation of the age of the early CL by ultrasonography improves the management of recipient mares in a commercial ET program

HCG is more effective than the GnRH agonist buserelin for induction of the first ovulations of the breeding season in sulpiride-primed recipient mares

In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using an artificial vagina during summer (December) and winter (July) and directly frozen. Subsequently, the frozen semen was thawed and analyzed by a computer-assisted semen analysis and flow cytometer assessing progressive motility, mean velocity, and DNA fragmentation spermatozoa. Kruskal–Wallis test and Pearson’s correlation were used to determine statistical differences among groups and correlation among variables (P ≤ .05). Both spermatozoa motility traits decreased progressively in accordance with age and seasonality, showing the lowest values in the aged group during winter and the highest values in the young group during summer. Deoxyribonucleic acid fragmentation increased significantly in accordance with age and seasonality being highest in the aged group during winter and lowest in the young group during summer. Post-thawing sperm quality showed a negative correlation with the age of the stallions and a positive correlation with the normal sperm morphology before freezing. These results allow the conclusion that age and seasonality are important factors that need to be considered during the selection of CPS for reproductive programs.

Transfer of equine in vitro produced blastocysts to recipient mares enables distinction of trophectoderm and inner cell mass subpopulations

Transfer of equine in vitro produced blastocysts to recipient mares enables distinction of trophectoderm and inner cell mass subpopulations

Effect of Supplementations with Saccharomyces cerevisiae and β-Glucans in Pregnant Recipient Mares on the Colostrum quality: Preliminary Results

Pythiosis is a life-threatening disease caused by the fungus-like microorganism Pythium insidiosum that can lead to death if not treated. Since P. insidiosum has particular cell wall characteristics, pythiosis is difficult to treat, as it does not respond well to traditional antifungal drugs. In our study, we investigated a new immunotherapeutic approach with potential use in treatment and in the acquisition of immunity against pythiosis. Dendritic cells from both human and mouse, pulsed with P. insidiosum heat-inactivated zoospore, (1,3)(1,6)-β-glucan and the immunotherapeutic PitiumVac® efficiently induced naïve T cell differentiation in a Th1 phenotype by the activation of specific Th1 cytokine production in vitro. Heat-inactivated zoospores showed the greatest Th1 response among the tested groups, with a significant increase in IL-6 and IFN-γ production in human cells. In mice cells, we also observed a Th17 pathway induction, with an increase on the IL-17A levels in lymphocytes cultured with β-glucan pulsed DCs. These results suggest a potential use of DCs pulsed with P. insidiosum antigens as a new therapeutic strategy in the treatment and acquisition of immunity against pythiosis.

Endogenous plasma progesterone levels in normal mares and embryo recipient mares between days 11 and 16 of gestation in a commercial breeding program

The present study was performed on 12 heads of donkey’s cadavers of both sexes for different anatomic techniques and on 20 live adult donkeys for ultrasonographic approaches of local anesthetic techniques. The aim was to achieve safe desensitization of the ear canal and tympanic membrane in addition to measuring different parameters of the structures occupying the infra-auricular parotid region. The internal auricular nerve was divided into two fine branches and constituting, at its origin, a characteristic V-shape with the caudal auricular nerve. The styloid process of auricular cartilage was an adequate landmark for ultrasonographic needle-guided anesthesia for internal auricular and auriculopalpebral nerves, whereas the great auricular nerve was easily palpated subcutaneously that showed safety and success in all cases. The auricular branch of mandibular nerve joined the auriculopalpebral branch of facial nerve. The maxillary vein was descending, partially embedded within the texture of the parotid glandular tissue. The parotid gland divided into five segments was clearly demarcated by maxillary vein tributaries with three main collecting radicles pouring into the parotid duct. The mandibular duct received about 12–15 fine radicles and supplied with a separate branch from the external carotid artery. Using the Doppler sonographic technique in donkeys for diagnosis of ear affections, evaluation of retrograde sialography to salivary glands with their blood vasculature and their indices might be helpful for the detection of different critical abnormalities, such as stenosis, thrombosis, and other vasopathological affections through measuring their resistivity and pulsatility indices.

A practical guide to estimate the age of the early CL by ultrasonography in mares examined for the first time to be used as recipients in a commercial embryo transfer program

The echogenicity of the early CL undergoes obvious changes during the first 5 days of luteal development. This phenomenon could potentially be used to estimate the age of the CL in mares which have not been checked frequently for the diagnosis of ovulation. The objective of this retrospective study was to determine the accuracy of estimating the age of the early CL (Day 0 to Day 4; Day 0 = Day of ovulation) in recipient mares examined for the first time in the breeding season to be used in a commercial embryo transfer (ET) program. The post-transfer pregnancy rate and embryo loss of 28 recipient mares whose CL age was estimated at the first scan of the breeding season (at least 2 months gap from the previous examination) were compared to that of 41 recipients in which the Day of ovulation had been determined by daily examinations and that received an embryo on the respective day. There was no difference (P > 0.1) in pregnancy (85.7 and 85.3%) or early embryonic loss (12.5% and 11.4%) between groups, respectively.

Clinical Application of in Vitro Embryo Production in the Horse

The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor mares (historically <25%; now >50%) and in the successful culture of zygotes to the blastocyst stage in vitro (historically <10%; now >20%). It has also been established that immature oocytes can be “held” at room temperature for at least 24 hours, allowing overnight transport to a laboratory with expertise in IVEP. Moreover, because in vitro–produced embryos can be cryopreserved with no appreciable reduction in viability, they can be shipped and stored until a suitable recipient mare is available for transfer. Most importantly, in an established equine ovum pick-up intracytoplasmic sperm injection (OPU-ICSI) program, blastocyst production rates now exceed 1 per procedure, and posttransfer foaling rates exceed 50%, such that overall efficiency betters that of either embryo flushing or oocyte transfer. Moreover, OPU-ICSI can be performed year round and allows embryo production from mares with severe acquired subfertility and extremely efficient use of scarce or expensive frozen semen. Cumulatively, these factors have stimulated rapid growth in demand for IVEP among sport horse breeders.