Assessing the fertility of two mares cloned from the same founder animal

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31–39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D₁₄), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p < .05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%—496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2–5-year-old control donor mares (same age of cloned mares) (89%—127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%—17/31), were both higher than for cloned mare (p < .05). The success rate of transfer was not different between embryos produced by cloned mares (84%—16/19) and those produced by control donor mares (79%—392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%—12/43) than for control donor mares (52% - 325/623) (p < .05).     

Surgical correction of rectovaginal fistula in mares and subsequent fertility

Objective To evaluate the fertility of mares bred at various intervals relative to surgical management of rectovaginal fistula (RVF). Materials and Methods Surgical repair of RVF was performed in 28 mares at variable times relative to foaling (30 days to 24 months) and also relative to rebreeding (same cycle or delayed). Postoperative fertility was then evaluated. Results Two mares were already pregnant at the time of surgery and 20 of 23 mares (87%) that were bred immediately prior to or following surgery conceived from their first service. When mares were bred in the same cycle as surgery, the next cycle following surgery or in the following breeding season after surgery the pregnancy rate was 5/5, 5/6 and 10/12, respectively, and the foaling rates were 4/5, 4/6 and 7/12. The two mares already pregnant at the time of surgery foaled successfully. Conclusions Excellent fertility can be achieved following surgical repair of RVF and our results suggest that delaying breeding until the following breeding season is not necessary. In addition, breeding in the same cycle as the surgical repair is a previously unreported technique that should be considered to maintain normal fertility and a yearly foaling interval.     

Surgical results and fertility following correction of vesicovaginal reflux in mares

Objective To evaluate the surgical results and fertility of mares bred at various intervals relative to surgical management of urovagina. Design Sixty-one mares underwent surgery to correct vesicovaginal reflux. Surgery was performed at varying times relative to breeding and postoperative fertility was evaluated. Results Five mares were lost to follow-up and a further four were electively not bred. Of the remaining 52 mares, seasonal pregnancy and live foaling rates were 67% (35/52) and 54% (26/48), respectively. When mares were bred in the same cycle as the surgery, the first cycle following surgery, the second cycle following surgery or the following breeding season after surgery the seasonal pregnancy rates were 89% (8/9), 63% (10/16), 67% (2/3) and 63% (15/24), respectively. After censoring four mares that died of natural causes pre-foaling, the foaling rates were 88% (7/8), 50% (7/14), 0% (0/3), 52% (12/23), respectively. All mares bred in the same cycle as surgery or in the next cycle were bred once only that season, so the pregnancy rate per cycle of 72% (18/25) was identical to the seasonal pregnancy rate. Conclusions Good fertility (comparable to a normal population of mares) was achieved following surgery and the results suggest that delaying breeding until the following breeding season is not necessary. In addition, breeding in the same cycle as surgical repair is a previously unreported technique that should be considered to maintain a yearly foaling interval.     

Studies of composition and major protein level in milk and colostrum of mares

The aim of the study was to determine the changes in composition and physicochemical features (pH, density, thermostability and acidity) of mare colostrum and milk, and of protein fraction contribution (serum albumin, β-casein, γ-casein, α-lactalbumin, G class immunoglobulins) depending on lactation stage. The research material was colostrum and milk samples from 12 Arabian mares. Colostrum samples were collected within 2 h after parturition and milk samples were collected twice, in the 3rd and 6th weeks of lactation. The level of basic milk components decreased significantly (only lactose content increased) as compared to colostrum. Total bacteria count and somatic cell count decreased significantly with an increase in resistance and urea level. The changes observed were connected to differentiated contribution of particular protein fractions and their relative proportions. Lower levels of γ-casein (P ≤ 0.05), β-casein, serum albumin as well as α-lactalbumin were observed in colostrum as compared to those in milk. Any relationship between lactation stage and β-casein content was observed. Serum albumin and α-lactalbumin content increased in subsequent milkings. The level of G class immunoglobulins decreased significantly and its highest level was noted in colostrum. Any significant differences between the 3rd and 6th lactation weeks were obtained.     

Analgesic, hemodynamic and respiratory effects of caudal epidurally administered ropivacaine hydrochloride in mares

Objective To determine the analgesic, hemodynamic and respiratory effects, sedation and ataxia in mares of caudal epidural administration of ropivacaine hydrochloride solution. Study design Prospective, single‐dose trial. Animals Ten healthy mares weighing from 475 to 565 kg. Methods Intravascular catheters and an epidural needle were placed after infiltration of the skin and subcutaneous tissues with 2% lidocaine. Ropivacaine (0.5%, 8 or 9 mL) was then injected epidurally at the fifth sacral or sacrococcygeal vertebrae, respectively. Analgesia was determined by lack of sensory perception to electrical stimulation (> 40 milliamps) and absence of response to needle pricks extending from coccyx to S2 dermatomes. Electrocardiogram, heart and respiratory rates, rectal temperature, arterial blood pressure, arterial acid‐base (pH, standard bicarbonate and base excess), gas tensions (PO₂, PCO₂), PCV, oxyhemoglobin and total solids concentrations, and numerical scores of perineal analgesia, sedation (head drop), and ataxia (position of pelvic limbs) were determined before and during a 5‐hour testing period. Analysis of variance (anova ) with repeated measures was used to detect significant (p < 0.05) differences of mean values from baseline. Results Epidurally administered ropivacaine induced variable analgesia extending bilaterally from coccyx to S2 (three mares), coccyx to S3 (four mares), and coccyx to S4 (three mares), with minimal sedation, ataxia, and cardiovascular and respiratory disturbances of mares. Perineal analgesia was attained at 10 ± 4 minutes and lasted for 196 ±42 minutes (mean ± SD). Five mares demonstrated inadequate perineal analgesia, probably attributable to deviation of the spinal needle from the midline. They were successfully blocked with ropivacaine on another occasion. Epidural ropivacaine significantly reduced repiratory rates of mares and did not change other variables from baseline. Conclusions and clinical relevance Ropivacaine (0.5%, 8 mL 500 kg^?1) can be administered caudal epidurally to produce prolonged (> 2.5 hours) bilateral perineal analgesia with minimal sedation, ataxia, and circulatory and respiratory disturbances in standing mares.     

The preliminary studies on protein profile in retained and not retained foetal membranes in heavy draft mares

Protein profile of the placenta expresses its function and maintenance. Any alterations can be reflected in qualitative and quantitative changes in this profile. The aim of the present study was the evaluation of protein profile in the placenta of mares suffering from the retention of foetal membranes (FMR) by two separation methods and the comparison with physiologically released tissues. Placentas from 14 healthy, heavy draft mares were collected immediately after the expulsion of newborn. Tissues after homogenization and staining with fluorescent dyes were subjected to electrophoretic as well as chromatographic separation. Electrophoretic gels were statistically analysed for the presence and abundance of examined proteins, while some proteins were identified in chromatographic fractions. Out of 248 spots detected in endometrium, 38 differed significantly between FMR and control animals, while in allantochorion, respective values reached 241 and 27 spots (p < .05). Among identified proteins that expressed higher abundance in endometrium of FMR mares than control animals were prostaglandin reductase, dehydrogenase/reductase SDR family, and placental growth factor. These proteins are involved in regulation of parturition. Additionally, the following proteins responsible for physiological activity of a cell—guanine methyl transferase, aspartyl/asparaginyl beta‐hydroxylase and GTP‐binding protein, were identified. These proteins expressed higher abundance in allantochorion of FMR mares than in controls. This preliminary study confirmed the disturbances in protein pattern between foetal membranes in FMR and healthy mares. Further qualitative and quantitative experiments are necessary to deepen our knowledge on the mechanisms of the retention of foetal membranes in mares.     

The nature of embryo reduction in mares with twin conceptuses: deprivation hypothesis

Ultrasonography was used to determine whether there is embryo reduction in mares with unilaterally fixed twins when a major portion of the vascularized area of the wall of one of the embryonic vesicles is in apposition with the wall of the adjacent vesicle, rather than with the endometrium (deprivation hypothesis). In addition, the effect of ovulatory pattern (synchronous and asynchronous) on the incidence of embryo reduction was studied. Twin vesicles were ultrasonically detected on days 11 to 15 (ovulation = day 0) and were examined daily until there was embryo reduction or until day 40. In 31 mares with twin embryonic vesicles, unilateral fixation (71%) was more frequent (P less than 0.05) than was bilateral fixation (29%). In 28 mares with known ovulatory patterns, synchronous ovulations did not affect the type of fixation (9/17 unilateral and 8/17 bilateral); however, for asynchronous ovulators the frequency of unilateral fixation (10/11) was greater (P less than 0.01) than the frequency of bilateral fixation (1/11). The incidence of embryo reduction was greater (P less than 0.01) for unilateral fixation (14/19) than for bilateral fixation (0/9) and was greater (P less than 0.05) for asynchronous ovulators (9/11) than for synchronous ovulators (5/17). In mares with embryo reduction, the reduction was complete before detection of both embryo propers (early reduction) in 10/14 and after detection of both embryo propers (late reduction) in 4/14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of short-term artificial light and transvaginal progesterone device on first ovulation in late transitional mares

In study I, plasma progesterone concentrations were evaluated in anoestrous mares that received an intravaginal progesterone release device (IPRD) for 10 days. Mares were divided into 3 groups based on the dosage of progesterone (0 g, n=3; 1.38 g, n=5; and 1.9 g, n=5). No statistical differences were found in plasma progesterone concentrations between the two doses tested. In study II, the effects of a protocol based on a short program of artificial light combined with an IPRD containing 1.38 g of progesterone on oestrous behaviour and onset of ovulation were evaluated. IPRDs were inserted into 31 late transitional mares (10 days of treatment). The mares were divided into a control group (n=9, IPRD with 0 g of progesterone) and two treatment groups (T1, n=10, IPRD with 0 g of progesterone and artificial light; T2, n=12, IPRD with 1.38 g of progesterone and artificial light). The percentages of mares in heat within the first 14 days after treatment were 100%, 70%, and 100% in the control, T1, and T2 groups, respectively (P=0.097), and their ovulation rates were 44%, 60%, and 100%, respectively (P≤0.01). In conclusion, a protocol based on artificial light and an IPRD containing 1.38 g of progesterone for 10 days could be considered to advance the first ovulation of the year in late transitional mares, as it ensures a higher rate of ovulation within the first 14 days after treatment.     

Equine amnionitis and fetal loss: The case definition for an unrecognised cause of abortion in mares

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.     

Comparative histomorphological study of endometrium in mares

Uterine acute post‐breeding inflammation is a physiological tissue response to the entry of exogenous elements, with persistent endometritis being the main pathology responsible for subfertility in the mare (Equus ferus caballus; Linnaeus, 1758). Mares can be classified as susceptible or resistant to endometritis according to their ability to remove intrauterine fluid within 48 hr after experimental inoculation. Endometrial biopsy is a technique that is commonly used to establish the degree of lesions that can affect the fertility of the mare. Endometrial histomorphometry is an objective and highly precise diagnostic method. The aim of this study was to compare, during oestrus, the endometrial histomorphometry of mares previously classified as susceptible (SM) or resistant (RM) to endometritis. Endometrial biopsies from 24 mares at the oestrus phase of the cycle were obtained. For the histomorphometric analysis, samples were histologically processed and subjected to routine Haematoxylin–Eosin staining. For the evaluation, the variables were considered as follows: 1‐Height of the lining and glandular epithelia (Lining SM = 15.9 μm vs. RM = 13.3 μm; Glandular SM = 15.0 μm vs. RM = 13.0 μm); 2‐Perpendicular diameters of endometrial glands (SM = 51.3 μm vs. RM = 44.8 μm); 3‐Number of endometrial glands per field (SM = 24.8 glands/field vs. RM = 20.5 glands/field). The results from this study suggest the existence of a relationship between the studied characteristics and the susceptibility/resistance to post‐breeding endometritis in mares. Thus, increased epithelial height, greater glandular density and greater development of the glands during oestrus would be related to a higher susceptibility to endometritis.     

Effect of oestrous synchrony between embryo donors and recipients,embryo quality and state on the pregnancy rate in beef cattle

This study aimed to evaluate the effect of oestrous synchrony between donors and recipients and the embryo quality on the pregnancy rate in beef cow recipients. The experiment was performed over two years at an embryo transfer (ET ) centre in Southern Brazil. Ninety Aberdeen Angus cows were subjected to superovulation (SOV ) protocols, resulting in the recovery of 1,048 transferable embryos. Eleven groups were formed with intervals of 6 hr, from ?30 to +30 hr, with respect to recipient versus donor oestrous detection. Evaluation of embryo quality was according to the IETS guidelines. The overall pregnancy rate was 52%. Effects related to donor and recipient oestrous synchronization on pregnancy rate were observed (p  =  .01), ranging from 36% to 50%. The embryo quality rate affected the pregnancy rate, where Grade I resulted in 57% and Grade III in 43% of pregnancy (p  <  .001). The embryonic state (frozen or fresh) showed no (p  >  .05) effect on pregnancy rate: 53% for fresh embryos and 44% for frozen embryos. The odds ratio for explanatory variables causing pregnancy indicated that Grade III embryos had 31% less chance of conception compared to Grade I. Thus, oestrous synchrony between donor and recipient, considering ±30 hr apart, can affect the pregnancy rate along with embryo quality.     

Folliculogenesis, embryo parameters and post-transfer recipient pregnancy rate following equine follicle-stimulating hormone (eFSH) treatment in cycling donor mares

Background Induction of multiple ovulations, or superovulation, may potentially increase the efficiency of equine embryo transfer programs. Our objective was to investigate the effects of equine follicle‐stimulating hormone (eFSH) treatment on the success rate of embryo transfer programs in mares. Methods In the research facility of the University of Saskatchewan, Canada, we studied 12 donor mares and 37 recipient mares during the physiological breeding season. Donor mares were used in two consecutive oestrous cycles: the first served as the control cycle and in the second an eFSH regimen was applied (eFSH cycle). In the control cycle, mares were administered human chorionic gonadotropin (hCG) to induce ovulation when a follicle ≥35 mm in diameter was detected by transrectal ultrasonographic examination. In the second oestrous cycle, twice‐daily eFSH treatment was initiated when a follicle ≥25 mm was detected and treatment ceased when a follicle ≥35 mm was present, at which time hCG was administered. All donor mares were artificially inseminated while in oestrus using fresh semen collected from a stallion of proven fertility. At 8 days post‐ovulation, embryos were recovered transcervically and transferred individually to the uterus of a synchronised recipient mare. Results The eFSH treatment stimulated the ovary and resulted in greater numbers of ovulations and recovered embryos; however the recovered embryos tended to have a lower morphological grade than the control embryos, and the recipient pregnancy rate per transferred embryo was lower than anticipated. Conclusion The numbers of recipient pregnancies and foals born that resulted from eFSH treatment were not different from the control.     

Embryo transfer in competition horses: Managing mares and expectations

Embryo transfer (ET) is an accepted and successful technique for obtaining foals from mares without interrupting their competition careers. Recent research, however, suggests that the potential of factors including heat, exercise, repeated embryo flushing and repeated manipulation of the reproductive cycle using exogenous hormones to have a negative impact on fertility may have been underestimated. This paper reviews the evidence base for involvement of these factors in repeated failures to recover embryos from nongeriatric competition mares without obvious clinical or pathological indications of reproductive abnormalities. It concludes that, for some mares at least, a cessation of exercise for the periovulatory period and the period between ovulation and embryo flushing, combined with careful management of flushing‐induced endometritis, and minimal hormonal manipulation of the reproductive cycle, may be necessary to optimise embryo recovery rates. Mare owners may have been encouraged to request ET for their mares following high‐profile examples in the media of elite mares that have produced foals by ET whilst competing. The veterinarian should educate mare owners about the multiple factors that may affect the chances of recovering an embryo from their mares, and should manage the expectations of mare owners so that they do not approach ET programmes in the expectation that there will be no disruption to their training and competition plans.     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

Achievements and future perspectives of embryo transfer technology in pigs

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non‐surgical ET and short‐ and long‐term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non‐surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short‐term use in pig production.     

复方中药“禽康散”对鸭瘟病毒感染鸭胚及成纤维细胞的保护效应

将不同浓度复方中药“禽康散”和鸭瘟病毒(Duck plague virus,DPV)分别以不同方式接种到鸭胚尿囊膜后观察其对DPV感染的鸭胚保护率;用MTT法测定“禽康散”对鸭胚成纤维细胞(DEF)的安全质量浓度和增殖的影响,及安全质量浓度范围内的“禽康散”对DPV感染鸭胚成纤维细胞(DEF)能力的影响.结果显示,“禽康散”在10日龄鸭胚内对DPV具有显著的杀灭和阻断感染的作用.“禽康散”对DEF的最大安全质量浓度为2-4 g/mL,在2-4～2-8 g/mL质量浓度范围内对DEF有显著的增殖和抑制DPV感染DEF的能力,且与加药方式和药物质量浓度有一定关系.结果表明,一定质量浓度的“禽康散”对DEF具有增殖作用,并对DPV在鸭胚内和细胞水平上均有较好的杀灭和拮抗作用.     

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8–9 and, in non-pregnant animals, basal concentration is reached around day 10–11 post-induction of ovulation. Luteolytic pulses of prostaglandin F_2α (PGF_2α) are firstly detected on day 7 or 8 (approximately on day 5–6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF_2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17β during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.     

Exogenous eFSH, follicle coasting, and hCG as a novel superovulation regimen in mares

The objective of this study was to evaluate various equine follicle-stimulating hormone (eFSH) treatment protocols and the effect of “follicle coasting” on ovulation and embryo recovery rates in mares. Cycling mares (n = 40) were randomly assigned to one of four groups 7 days after ovulation: (1) 12.5 mg eFSH twice daily until follicles were 35 mm or larger; (2) 12.5 mg eFSH twice daily until follicles were 32 mm or larger; (3) 12.5 mg eFSH twice daily for 3.5 days followed by 12.5 mg eFSH enriched with luteinizing hormone (LH) twice daily until follicles were 35 mm or larger; and (4) 25 mg eFSH once daily until follicles were 32 mm or larger. Mares in groups 1 and 3 were injected with human chorionic gonadotropin (hCG) (2500 IU intravenously) at the end of eFSH treatment, whereas mares in groups 2 and 4 were given hCG approximately 42 and 54 hours, respectively, after the last eFSH treatment (“follicle coasting”). Nonsurgical embryo collection was performed 6.5 to 7.5 days after ovulation. Each mare experienced a nontreated estrous cycle before being reassigned to a second treatment. Ovulation rates for mares in treatment groups 1 to 4 were 3.3 ± 0.4, 4.1 ± 0.4, 3.5 ± 0.4, and 2.8 ± 0.4 (mean ± SEM; P < .05), respectively. One or more embryos were recovered from more than 80% of mares in each treatment group, and embryo recovery rate per flush was similar among treatment groups (1.9 ± 0.3, 2.6 ± 0.3, 1.9 ± 0.3 and 1.9 ± 0.3, respectively; P > .05). The overall embryo recovery rate was 2.1 ± 1.5 embryos per flush. In summary, ovulation rate was higher for mares treated with eFSH (3.4 ± 0.4) compared with non-treated controls (1.1 ± 0.2). Ovulation rate in mares in which hCG was delayed (follicle coasting) was higher (P < .05) when treatments were given twice per day versus once per day. Administration of equine luteinizing hormone (eLH) in conjunction with eFSH did not have an advantage over mares treated only with eFSH.