Successful Non-Surgical Transfer of Horse Embryos to Mule Recipients

Mules, hybrids resulting from the mating of a horse mare (Equus caballus, 2n = 64) to a Jack donkey (E. asinus, 2n = 62), are generally infertile. Five horse embryos were transferred non‐surgically to two cyclic and one acyclic recipient mules. In the mares and cycling mules, oestrus and ovulation were induced with, respectively, d ‐cloprostenol and human chorionic gonadotrophin (hCG). The acyclic mule, on the other hand, received oestradiol benzoate when the embryo donor was showing oestrus and progesterone after the donor had ovulated and until pregnancy diagnosis. Non‐surgical embryo collections were attempted on day 7 after ovulation and recovered embryos were transferred transcervically into the mules’ uteri. Mules that became pregnant were blood sampled serially for equine chorion gonadotrophin (eCG), progestagen and total conjugated oestrogen concentrations until around 6 months of gestation. The three embryos transferred to the acyclic mule did not produce any pregnancies whereas both embryos transferred to the cycling mules resulted in the birth of live foals. The peak concentration and duration of secretion of eCG differed markedly between the two pregnant mules, although both animals appeared to develop secondary corpora lutea beyond day 40 of gestation, as in normal intraspecies horse pregnancy. Moreover, the rise in serum oestrogen concentrations from around day 90 was also similar to that seen in normal pregnant mares. Parturition occurred spontaneously on day 348 of gestation in both mules and the resulting colt foals developed normally to weaning. Thus, cycling mules can carry a horse conceptus after non‐surgical embryo transfer and give birth to a normal mature foal.     

The effects of an advanced uterine environment on embryonic survival in the mare

Reasons for performing the study: During embryo transfer (ET) the equine embryo can tolerate a wide degree of negative asynchrony but positive asynchrony of >2 days usually results in embryonic death. There is still confusion over whether this is due to the inability of the embryo to induce luteostasis or to an inappropriate uterine environment. Objectives: To assess embryo survival and development in an advanced uterine environment. Hypothesis: Embryo–uterine asynchrony, not the embryo's inability to induce luteostasis, is responsible for embryonic death in recipient mares with a >2 days chronologically advanced uterus. Methods: Experiment 1: Thirteen Day 7 embryos were transferred to the uteri of recipient mares with luteal prolongation, occasioned by manual crushing of their own conceptus, such that donor–recipient asynchrony was between +13 and +49 days. Experiment 2: Day 7 embryos were transferred to recipient mares carrying their own conceptus at Days 18 (n = 2), 15 (n = 2), 14 (n = 4), 12 (n = 4) or 11 (n = 4) of gestation. In addition, Day 8 embryos were transferred to 4 pregnant recipient mares on Day 11 of gestation. Results: No pregnancies resulted following transfer of Day 7 embryos to recipients in prolonged dioestrus with asynchronies between +13 and +49 days. However, the use of early pregnant mares as recipients resulted in 5/20 (25%) twin pregnancies, 4 of which came from the transfer of a Day 8 embryo to a Day 11 recipient. All transferred embryos showed retarded growth, with death occurring in 4/5 (80%). Conclusions and potential relevance: The results emphasise the importance of an appropriate uterine environment for embryo growth and the inability of equine embryos to survive transfer to a uterus >2 days advanced even when luteostasis is achieved. It is possible that in normal, non‐ET equine pregnancy, embryo–uterine asynchrony may account for some cases of embryonic death.     

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well‐being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well‐being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.     

Progesterone and equine chorionic gonadotropin concentrations around the time of pregnancy loss in mares impregnated by donkeys or stallions

The objective of this study was to compare the incidence of pregnancy loss of mares carrying a mule embryo with that of mares carrying a horse embryo. The possible causes of such mortality were evaluated through serial ultrasonographic evaluations and hormonal monitoring, paying special attention to the role of premature regression of the endometrial cups and its relation to inadequate luteal function.

Twenty-eight mares impregnated by stallions and 19 mares impregnated with donkey semen were evaluated ultrasonographically every week from day 20 to day 150 of pregnancy. The viability of the product was assessed each time, and the diameter of the embryonic vesicle was measured from day 25 to day 60. Blood samples for progesterone and equine chorionic gonadotropin (eCG) determination were taken every week.

Both progesterone and eCG concentrations during normal pregnancies wegre significantly lower in the mares inseminated with donkey semen than in the mares impregnated by stallions (P<.05). In 7 of the mares carrying mule conceptuses to term, the concentrations of eCG remained basal throughout the study. In the other animals from this group, the levels of this hormone did increase but returned to baseline much earlier (on day 77 of pregnancy) than in the mares served by stallions (on day 126 day of pregnancy). There was no significant difference between the growth rate of embryonic vesicles of mares carrying mule embryos and that of mares carrying horse embryos (P>.05).

The incidence of pregnancy loss was significantly higher (P<.05) in mares carrying a mule embryo (36.8 %) than in mares carrying a horse embryo (21.4%); it occurred on average on day 93 of pregnancy in mares carrying mule embryos and on day 43 on mares carrying horse embryos. There was only 1 case in which pregnancy loss was associated with concentrations of both eCG and progesterone that were much lower than the average for the normal pregnancies of the same group, and this was in a mare carrying a horse embryo. The most frequent cause of pregnancy loss was premature luteal regression due to primary luteolysis, as evaluated via peripheral progesterone concentrations. This occurred in 2 mares carrying horse embryos and in 4 mares carrying mule embryos. Three mares carrying mule embryos and 1 carrying a horse embryo had abortions that were not preceded or accompanied by any alteration in progesterone or eCG levels and were thus classified as fetal deaths of non-endocrine origin.

It is concluded that the incidence of pregnancy loss is higher in mares carrying a mule embryo than in mares carrying a horse embryo. However, this is not due to the low progesterone concentrations associated with the premature regression of the endometrial cups that occurs in mares with interspecific pregnancy.     

Season Does Not Influence Embryo Recovery Rate and Conceptus Size Until Day 14 After Ovulation in the Horse

Although the horse is a seasonal breeding species, a considerable number of mares continue to cycle throughout autumn and winter. Slower equine embryo growth during the non‐breeding season has been hypothesized, and because smaller embryo size is beneficial for cryopreservation, embryo collection outside the breeding season could be an interesting approach for the production of frozen horse embryos. In the present retrospective study, we have therefore analysed embryo recovery rates and conceptus size in mares (n = 30) throughout the year. Conceptus diameter was either size determined after collection with a microscopic scale (day 7–10 after ovulation) or determined by transrectal ultrasound immediately before collection (day 11–14 after ovulation). In 19 of the 30 mares (63%), ovulatory cycles were detected throughout the year. A total of 352 embryo collections with a mean recovery rate of 64.2% were performed and not affected by season. The size was analysed in a total of 165 conceptuses. Conceptus diameter significantly increased (p < 0.001) with day of pregnancy (e.g. day 7: 0.3 ± 0.04, day 10: 4.1 ± 0.2, day 12: 10.1 ± 0.5, day 14: 17.4 ± 0.9 mm), but was not influenced by season. In conclusion, successful embryo collection is possible throughout the year in spontaneously cyclic mares. Under these conditions, neither collection rates nor embryo growth appeared to be affected by season.     

Practical Experience with the Treatment of Recipient Mares with a Non‐Steroidal Anti‐Inflammatory Drug in an Equine Embryo Transfer Programme

As part of a commercial embryo transfer programme, 20 embryos were transferred to spontaneously synchronous or synchronized recipient mares. In 14 cases, embryo recipients were treated with non‐steroidal anti‐inflammatory drugs (NSAID), receiving flunixin meglumine i.v. at the time of transfer and vedaprofen orally twice a day on the 3 days after embryo transfer, while six embryos were transferred to untreated mares that served as controls. Out of the 14 recipient mares treated with NSAID, 11 (79%) were pregnant at 6–8 days after transfer and in 10 mares, the pregnancy was continued. From the six untreated recipients, only one became pregnant but underwent early embryonic death between day 14 and 35 after ovulation. In conclusion, pregnancy rate in NSAID‐treated recipients is higher than that in untreated recipients and above reported average values, indicating that treatment of recipient mares with NSAID helps to increase pregnancy rates after transcervical transfer and can be recommended for equine embryo transfer.     

Luteoprotective Role of Equine Chorionic Gonadotropin (eCG) During Pregnancy in the Mare

The effects of repeated cloprostenol administration were compared in mares impregnated by horses and mares impregnated by donkeys in order to assess the role of eCG on the development of pregnancy‐associated resistance to the luteolytic and abortifacient effects of PGF2α. Eleven mares impregnated by donkey (mule pregnancy) and 9 mares impregnated by horse (horse pregnancy) were used. Six mares with mule pregnancy and four with horse pregnancy were injected with cloprostenol (0.25 mg) when they were between day 65 and day 75 of pregnancy, and the treatment was repeated 48, 72 and 96 h latter. The rest of the mares remained as controls. Concentrations of eCG were 10 times higher (p < 0.001) in mares impregnated by horses than in mares impregnated by donkeys, and they were not affected by cloprostenol treatment. Luteolysis was completed 30 h after the first cloprostenol injection in mule pregnancies, while mares with horse pregnancies required 96 h and three cloprostenol injections to complete luteolysis. Regression analysis revealed significant associations between eCG concentrations at time 0 and the time required for completion of luteolysis (p < 0.001), foetal death (p < 0.01) and foetal expulsion (p < 0.05). It is concluded that high eCG concentrations in mares impregnated by horses protect the corpora lutea of pregnancy against the luteolytic effects of PGF2α. Low eCG concentrations in mares carrying mule foetuses afford them less protection against the luteolytic effect of PGF2α, and this may be a cause of the increased foetal mortality that occurs between days 60 and 90 of pregnancy in these mares.     

Luteal vascularity and embryo dynamics in mares during early gestation: Effect of age and endometrial degeneration

The present study characterized the luteal status and the dynamic of the conceptus during the first 20 days of gestation in mares with different ages and degrees of endometrial degeneration. Total area of the corpus luteum (CL), luteal vascularity, CL area with blood signals, progesterone concentrations (P4), embryonic vesicle diameter, number of embryonic location changes, embryonic fixation position and uterine contractility were evaluated. In Experiment 1, mares ≤6 years of age (Young group, 5.6 ± 0.2 years, n = 7 mares) and mares ≥15 years of age (Old group, 17.2 ± 0.9 years, n = 6 mares) were used to investigate the effect of age. In Experiment 2, the luteal and embryonic parameters were compared between mares with minimal (Mild group, endometrial category I, n = 9 mares) and severe (Severe group, endometrial category III, n = 7 mares) endometrial degeneration. The Old and Severe groups had greater (p ≤ 0.04) total CL area and reduced luteal vascularity (p ≤ 0.04) than the Young and Mild groups, respectively. However, P4 levels and CL area with blood signals were similar (p ≥ 0.8) between the groups. A negative effect of age (p < 0.01), but not of endometrial degeneration (p = 0.6), was found for the embryonic vesicle diameter. The conceptus mobility was high (p > 0.1) until day 14 of gestation in the Severe group, while a reduced number of changes of the embryo location was detected earlier (p < 0.05) in the Old group. In conclusion, the newly formed CL of aged mares and mares with severe endometrial degeneration suffered a structural remodelling to safeguard the local blood supply and the continuous P4 output during early gestation. Moreover, an earlier reduction of the embryonic mobility and a delayed development of the conceptus were associated with advanced age, regardless of the degree of endometrial degeneration.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

Using “Uterine Index” to diagnose embryonic death in mares

Embryonic death plays an important role in infertility in mares, and most of embryonic mortality occurs at an early stage (before day 40) of pregnancy. Previous investigations focused on B-mode sonographic images to determine abnormal pregnancy; however, no study has provided a quantitative and objective method to diagnose embryonic death. Here we developed a new index, “Uterine Index,” using color Doppler sonography to describe alterations of uterine blood flow in physiological and pathological gestations during early gestation. Six Trotter mares were used in this study. The pregnancies were interrupted with a prostaglandin F2α (PGF_2α) analog (tiaprost) at days 25, 30, and 40. Three gestations were investigated in each stage. After the PGF_2α administration, these mares were examined every 8 hours until the embryonic vesicles were not visible on ultrasound. In the control group, three pregnancies were examined daily from days 12 to 60. The embryo size was measured in B-mode sonography, and the blood flow in uterine arteries was examined in Doppler ultrasound. The resistance index (RI) and uterine index (UI) were calculated. In the control group, the RI values of uterine artery were lower in pregnant side compared with that of the non-pregnant side from day 15 on, and the difference became greater as the pregnancy advanced (P < .05). The UI was close to 0 before week 3 and increased to approximately 10 after week 4. In the PGF_2α-treated group, the UI values reduced from pregnancy level to approximately 0. As early as 32 hours before embryonic death, we observed a significant difference of UI values between control and treated mares. On the contrary, the embryo size of treated mares was not different from that of normal mares. Through the analysis of all recorded UI values, we suggest that the UI values 10 and 5 are good values for distinguishing normal pregnancy (UI >10) from embryonic death (UI < 5). If the UI values are located between 10 and 5, a reexamination at intervals of 1 to 3 days is recommended. Our findings demonstrate that the UI is a useful method to diagnose and predict embryonic death in mares by providing reliable and objective information.     

Some Factors Affecting the Rate of Pregnancy after Embryo Transfer Derived from the Brazilian Jumper Horse Breed

This study aimed to evaluate the effects of certain embryo transfer parameters on the pregnancy rate after equine embryo transfer of the Brazilian Jumper Horse breed. The size, embryonic development stage, embryo quality, and synchronization of ovulation between the donor (n = 120) and recipient (n = 420) were evaluated in 396 embryos. Embryo recovery was performed on Day 6-9 after ovulation (Day 0 = day of ovulation). The recipient mares were chosen on the day of embryo recovery, and the transfers were performed that same day. The embryo size (diameter including envelopes; n = 396) ranged from 150 to 3000 μm; 67.1% measured between 400 and 1199 μm. The embryo size (400-1199 μm vs. ≤399 μm); stage of development (n = 396; blastocyst and expanded blastocyst versus compact morula and early blastocyst); quality (n = 396; grade 1 [excellent]), 2 [good], or 3 [poor]); and synchronization of ovulation between the donor and recipient (0, 1, 2, 3, and 4 days versus −1, 5, and 6 days, respectively) all affected pregnancy rate (P < .05). The pregnancy rate did not differ significantly among transfers performed on Days 0, 1, 2, 3, and 4. In conclusion, embryos measuring 400-1199 μm produced higher pregnancy rates in recipients than embryos measuring 150-399 μm, and blastocysts and expanded blastocysts produced pregnancy more efficiently than morulae and early blastocysts. The embryo quality also affected the pregnancy rate. Synchronization of donor and recipient ovulation to Days 0-4 improved the efficiency of embryo transplant.     

Key Factors Affecting Reproductive Success of Thoroughbred Mares and Stallions on a Commercial Stud Farm

To evaluate factors contributing to fertility of thoroughbred mares, data from 3743 oestrous periods of 2385 mares were collected on a large thoroughbred farm in Ireland. Fourteen stallions (mean age 8.3 years; range 4–15 years) had bred 2385 mares (mean age 9.4 years; range 3–24 years). Maiden mares accounted for 12%, mares with a foal at foot for 64%, and barren, slipped or rested mares for 24% of the total. The mean pregnancy rate per cycle was 67.8% (68.6% in year 1 and 66.9% in year 2). Backward stepwise multivariable logistic regression analysis was utilized to develop two models to evaluate mare factors, including mare age, reproductive status, month of foaling, dystocia, month of cover, foal heat, cycle number, treatments, walk‐in status and stallion factors including stallion identity, stallion age, shuttle status, time elapsed between covers and high stallion usage on the per cycle pregnancy rate and pregnancy loss. Old age (p < 0.001) and cover within 20 days post‐partum (p < 0.003) were associated with lowered pregnancy rates. High mare age (p < 0.05) and barren, slipped or rested reproductive status (p = 0.05) increased the likelihood of pregnancy loss. Uterine inflammation or infection, if appropriately treated, did not affect fertility. Only high usage of stallions (used more than 21 times in previous week) was associated with lowered (p = 0.009) pregnancy rates. However, shuttle stallions were more likely to have increased (p = 0.035) pregnancy survival, perhaps reflecting a bias in stallion selection. In conclusion, mare age exerted the greatest influence on fertility; nonetheless, thoroughbreds can be effectively managed to achieve high reproductive performance in a commercial setting.     

A Field Study of Serum,Colostrum, Milk Iodine,and Thyroid Hormone Concentrations in Postpartum Draft Mares and Foals

Iodine, thyroxine (T4) and triiodothyronine (T3) are required for normal fetal growth, maturation, and neonatal survival. There is a lack of robust information on iodine levels found in colostrum, milk, and serum of mares and foals after a healthy pregnancy. Our objective was to characterize colostrum, milk, and serum iodine levels in healthy postpartum mares and foals (n = 10) and explore relationships with thyroid hormone concentrations. Colostrum, milk, and jugular blood samples from draft breed mares and foals with an estimated average iodine daily intake of 39 mg per mare during pregnancy were obtained at Day 0 (foaling date) and/or 10 days later. Parameters studied were (1) mare basal concentrations of serum: TT3, TT4, and iodine; (2) iodine in colostrum at Day 0 and milk iodine (Day 10); and (3) foal basal: TT3, TT4, and serum iodine (Days 0 and 10). Median ± median error colostrum iodine levels (165 ± 15.1 μg/L) were higher than milk (48 ± 5.6 μg/L; P = .007) levels. Median ± median error foal serum iodine (268.5 ± 7.6 μg/L), TT4 (1,225 ± 47.8 nmol/L), and TT3 (14.2 ± 1.1 nmol/L) at foaling date were higher than at 10 days (serum iodine: 70 ± 3.6 μg/L; TT4: 69.6. ± 20.4 nmol/L; and TT3: 5.4 ± 0.3 nmol/L). In conclusion, equine mammary tissue concentrates iodine beyond plasma levels, making colostrum and milk a significant source of iodine. Foal serum iodine levels are high in the neonatal period and are positively correlated with TT4, which is important for neonatal adaptation.     

Serial investigations of early pregnancy in pony mares using real time ultrasound scanning

The uteri of 13 pony mares were examined daily by ultrasound during the first two months of gestation. The conceptus was first identified between 12 and 16 days after ovulation and the embryo was seen on the ventral surface of the conceptus after Day 21. The foetal heart could be visualised after Day 22 and spontaneous movement of the foetus occurred after Day 39. No consistent pattern was seen in the development of the foetal membranes, although attachment of the umbilical cord to the allantochorion was always on the dorsal aspect of the conceptus. Daily measurements were made of the diameter of the conceptus and the length of the embryo and foetus.     

Diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares

OBJECTIVE: To determine the incidence, ultrasonographic characteristics, and risk factors associated with embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. DESIGN: Prevalence survey. ANIMALS: 159 pregnant mares. PROCEDURES: From 1994 to 1998, mares between 11 and 40 days after ovulation with normal and abnormal embryonic development were examined ultrasonographically, and characteristics of each conceptus were recorded. RESULTS: The incidence of abnormal embryonic development in mares characterized by formation of an embryonic vesicle without an embryo was 7/159 (4.4%) during the 5 breeding seasons. Age and breed of mare or type of semen used did not differ for mares with normal and abnormal embryonic development. The percentage of mares in which the conceptus was undersized during > or = 1 examination was significantly higher for mares with abnormal conceptuses (5/7), compared with mares with normal conceptuses (2/147; 1.4%). The percentage of examinations during which the conceptus was undersized was significantly higher for abnormal conceptuses (12/27; 44.4%), compared with normal conceptuses (4/448; 0.9%). CONCLUSIONS AND CLINICAL RELEVANCE: To diagnose an embryonic vesicle without an embryo, mares should be examined by use of transrectal ultrasonography on day 25 after ovulation. When an embryo cannot be identified at that time, mares should be reexamined at intervals of 1 to 3 days until day 30. Because undersized conceptuses are more likely to be abnormal, development of undersized conceptuses should be monitored closely.     

Factors influencing equine chorionic gonadotrophin production in the mare

Reasons for performing study: An opportunity to monitor equine chorionic gonadotrophin (eCG) production during 61 pregnancies in 25 Thoroughbred mares mated to the same Thoroughbred stallion was utilised in order to further knowledge regarding factors involved in the production of this hormone. Objectives: To examine the effects of maternal body condition, exercise and parity on eCG production. Methods: In the first experiment, maiden mares were fed either a moderate (n = 9) or an excessive (n = 10) food intake throughout gestation. In the second experiment, 5 mares were exercised daily during pregnancy and eCG production rates were compared to 5 nonexercised mares. In the third experiment, eCG profiles were compared in 9 mares during 3 successive pregnancies. Equine chorionic gonadotrophin secretion was assessed as area under the curve (AUC), peak serum concentration, timing of the peak and the rate of decline. In addition, a mean eCG profile of 61 pregnancies was created to provide means and ranges for the above parameters. Results: In Experiment 1, eCG production was significantly higher in moderately rather than excessively fed mares in terms of AUC and peak eCG concentrations. In Experiment 2, the mean AUC did not differ between exercised and nonexercised animals but mean eCG concentrations were significantly higher in nonexercised mares between Days 60 and 90 of gestation. In Experiment 3, eCG became undetectable significantly earlier in gestation in the third parity. The mean eCG profile of 61 pregnancies showed a peak of 64.5 ± 3.7 iu/ml at 62.4 ± 1.0 days after ovulation and was undetectable by 134.1 ± 1.7 days. Peak eCG levels reduced by 50% 22.6 ± 1.13 days. Conclusions: Some of the factors examined clearly influenced eCG production rate, the secretion of this hormone and its rate of disappearance from the blood. Potential relevance: The results provide insights into some factors that govern the production of the placental gonadotrophin, eCG.     

Metabolomic profiling in umbilical venous plasma reveals effects of dietary rumen‐protected arginine or N‐carbamylglutamate supplementation in nutrient‐restricted Hu sheep during pregnancy

Maternal nutrient restriction during pregnancy is a major problem worldwide for human and animal production. Arginine (Arg) is critical to health, growth and reproduction. N‐carbamylglutamate (NCG), a key enzyme in arginine synthesis, is not extensively degraded in rumen. The aim of this study was to investigate ameliorating effects of rumen‐protected arginine (RP‐Arg) and NCG supplementation on dietary in undernourished Hu sheep during gestation. From day 35 to 110 of gestation, 32 Hu ewes carrying twin foetuses were randomly divided into four groups: a control (CG) group (n = 8; fed 100% National Research Council (NRC) requirements for pregnant sheep), a nutrient‐restricted (RG) group (n = 8; fed 50% NRC requirements, which included 50% mineral–vitamin mixture) and two treatment (Arg and NCG) groups (n = 8; fed 50% NRC requirements supplemented with 20 g/day RP‐Arg or 5 g/day NCG, which included 50% mineral–vitamin mixture). The umbilical venous plasma samples of foetus were tested by ¹H‐nuclear magnetic resonance. Thirty‐two differential metabolites were identified, indicating altered metabolic pathways of amino acid, carbohydrate and energy, lipids and oxidative stress metabolism among the four groups. Our results demonstrate that the beneficial effect of dietary RP‐Arg and NCG supplementation on mammalian reproduction is associated with complex metabolic networks.     

Surgical Removal of One Conceptus from Fifteen Mares with Twin Concepti

A surgical technique for removal of one conceptus from mares with twin concepti more than 35 days of gestational age was evaluated. One conceptus was removed surgically from each of 15 mares carrying twin concepti that were 41 to 65 days of gestational age. As determined by ultrasonography, eight mares had bicornuate and seven mares had unicornuate twin concepti. For maintenance of pregnancy if surgical trauma should cause prostaglandin release and luteolysis, progesterone was administered prophylactically. Flunixin meglumine was administered perioperatively to minimize prostaglandin release. Five mares with bicornuate twin concepti delivered a single viable foal; in one mare the fetus was alive 4 days after surgery when the mare was euthanatized because of a fractured femur. None of the remaining feti in the seven mares with unicornuate twin concepti survived. The poor survival rate of unicornuate twin concepti was attributed to disruption of the remaining chorioallantois during surgery. Thirteen mares have been rebred successfully.     

Ovulation,Pregnancy Rate and Early Embryonic Development in Vernal Transitional Mares Treated with Equine‐ or Porcine‐FSH

The objective of this study was to compare the efficacy of purified equine‐ and porcine‐FSH treatment regimes in mares in early vernal transition. Mares (n = 22) kept under ambient light were examined ultrasonographically per‐rectum, starting January 30th. They were assigned to one of two treatment groups using a sequential alternating treatment design when a follicle ≥ 25 mm was detected. In the eFSH group, mares were treated twice daily with equine‐FSH, and in the pFSH group mares were treated twice daily with porcine‐FSH; treatments were continued until follicle(s) ≥ 35 mm, and 24 h later hCG was administered. Oestrous mares were inseminated with fresh semen and examined for pregnancy on days 11–20 post‐ovulation. In the eFSH group, 11/11 (100%) mares developed follicle(s) ≥ 35 mm, 8/11 (73%) ovulated and 6/8 (75%) conceived. In the pFSH group, 5/11 (45%) developed follicle(s) ≥ 35 mm, 4/11 (36%) ovulated and 3/4 (75%) conceived. Treatment with eFSH resulted in a greater ovarian stimulation; higher number of pre‐ovulatory‐sized follicles, higher number of ovulations and higher number of embryos (p < 0.05). Following ovulation, serum progesterone concentrations were correlated with the number of CLs and supported early embryonic development; maternal recognition of pregnancy occurred in all pregnant mares. We concluded that eFSH can be used to effectively induce follicular growth and ovulation in vernal transitional mares; however, if bred, diagnosis and management of twins’ pregnancies would be required prior to day 16 because of the increased risk of multiple embryos per pregnancy. Conversely, the current pFSH treatment regime cannot be recommended.     

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.