The post-ovulatory rise in progesterone is lower and the persistence of oestrous behaviour longer during the first compared with the second cycle of the breeding season in mares.

Mares are seasonally polyoestrous breeders. Therefore, the first ovulation of the season, following winter anoestrus, is the only cycle in which mares ovulate without the presence of an old CL from the previous cycle. The objective of this study was to compare the length of oestrous behaviour, and plasma progesterone concentrations during the early post-ovulatory period between mares after the first and second ovulation of the breeding season. Overall, 38 mares and 167 oestrous periods were used in the study. From those, 11 mares were used during the first and subsequent oestrous period to measure and compare the post-ovulatory rise in progesterone concentration, whereas all the mares were used to compare the length of the post-ovulatory oestrous behaviour between the first and subsequent cycles of the breeding season. The persistence of the post-ovulatory oestrus was longer (p < .001) following the first ovulation of the year (median of 52 h) compared with the subsequent ovulations (median of 36 h for second and later ovulations groups; n = 38 mares). The progesterone concentration at any of the four 8 h-intervals analysed (28, 36, 76 and 84 h post-ovulation) was lower (p < .01) following the first versus the second ovulation of the year. By 36 h post-ovulation the progesterone concentration of mares at the second ovulation of the year had passed the threshold of 2 ng/ml (2.1 ± 0.33 ng/ml), whereas in the first cycle it was 1.2 ± 0.13 ng/ml. In conclusion, mares had lower progesterone concentrations in their peripheral circulation and longer persistence of oestrous behaviour following the first ovulation of the year compared with the second and subsequent ovulatory periods of the breeding season.

A preliminary study on the glycosylation of the reproductive tract in the Ostrich (Struthio camelus camelus).

Glycosylation of the reproductive tract of an adult female red-necked ostrich (Struthio camelus camelus) carrying a fully formed calcified egg in her uterus when accidently killed by a blow to the head was examined using lectin histochemistry on samples from the infundibulum, magnum, uterus and vagina. Glycans in the luminal epithelium and underlying glands were described after staining with 23 lectins after neuraminidase pre-treatment in some cases. Ciliated and non-ciliated cells were evident at all levels in the luminal epithelium, the latter full of richly glycosylated secretory granules. The ciliated cells also showed glycosylation and, in the magnum, these cells often stained more intensely than the non-ciliated cells. High mannose and complex N-glycans, α1,6-linked fucosyl and sialic acid residues were present throughout the tract and there was a complete absence of GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1- and rare terminal GalNAcα1- residues. Fucose in α1,2-linkage as H2 antigen and Ley was also rare in the luminal epithelium and completely absent in glands. Terminal galactose was present in the luminal epithelium apart from in the infundibulum. Gland epithelium showed similar glycosylation to the luminal epithelium except in the magnum where there were significant differences and here the glands were packed full of large secretory granules, unlike the glands in the rest of the tract. Each section of the tract had its own specific pattern of glycosylation which could be related to the stage of egg formation.

Placentation in the plains zebra (Equus quagga).

The placenta and fetal gonads of 12 pregnant plains zebra (Equus quagga), estimated to be between 81 and 239 days of gestation, were examined. The diffuse, microcotyledonary zebra placenta appeared, developmentally, to be 3-4 weeks behind its counterpart in horse pregnancy and this, together with the presence of small and long-lived endometrial cups, low levels of zebra chorionic gonadotrophin in maternal serum and few accessory corpora lutea in the maternal ovaries during the first half of gestation, made zebra pregnancy more similar to donkey than horse pregnancy. Zebra fetal gonads enlarged after 80 days of gestation and their interstitial cells stained positively for 3ß hydroxysteroid dehydrogenase and 17,20 lyase steroid enzymes while the trophoblast stained for aromatase. This confirmed that zebra fetal gonads, like those of the horse and donkey, can synthesise C19 androgens, which can then be aromatised by the placenta to C18 oestrogens. It is remarkable that such unusual feto-placental mechanisms of production of gonadotrophic and steroid hormones has persisted unchanged within the genus Equus despite the many physical adaptations and the considerable loss of chromosomes that have occurred during the evolution of its member species.

The binucleate cell of okapi and giraffe placenta shows distinctive glycosylation compared with other ruminants: a lectin histochemical study.

The placenta of ruminants contains characteristic binucleate cells (BNC) with a highly conserved glycan structure which evolved early in Ruminant phylogenesis. Giraffe and Okapi placentae also contain these cells and it is not known whether they have a similar glycan array. We have used lectin histochemistry to examine the glycosylation of these cells in these species and compare them with bovine BNC which have a typical ruminant glycan composition. Two placentae, mid and near term, from Giraffe (Giraffa camelopardalis) and two term placenta of Okapi (Okapia johnstoni) were embedded in resin and stained with a panel of 23 lectins and compared with near-term bovine (Bos taurus) placenta. Significant differences were found in the glycans of Giraffe and Okapi BNC compared with those from the bovine, with little or no expression of terminal αN-acetylgalactosamine bound by Dolichos biflorus and Vicia villosa agglutinins which instead bound to placental blood vessels. Higher levels of N-acetylglucosamine bound by Lycopersicon esculentum and Phytolacca americana agglutinins were also apparent. Some differences between Okapi and Giraffe were evident. Most N-linked glycans were similarly expressed in all three species as were fucosyl residues. Interplacentomal areas in Giraffe and Bovine showed differences from the placentomal cells though no intercotyledonary BNC were apparent in Okapi. In conclusion, Giraffidae BNC developed different glycan biosynthetic pathways following their split from the Bovidae with further differences evolving as Okapi and Giraffe diverged from each other, affecting both inter and placentomal BNC which may have different functions during development.

Successful vitrification of equine embryos >300 microns without puncture or aspiration.

BACKGROUND: Equine embryos >300 µm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use. OBJECTIVES: To design a successful vitrification protocol for embryos >300 µm without puncture as a pre-treatment. STUDY DESIGN: Experimental in vivo study. METHODS: Thirty-eight embryos were divided into 3 groups (G1: ≤300 µm, n = 11; G2: >300-500 µm, n = 20; G3: >500 µm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient. RESULTS: Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 µm. MAIN LIMITATIONS: Limited numbers and only one pregnancy was followed to term. CONCLUSIONS: Equine embryos ≤480 µm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development.

Persistence of an immunoreactive MUC1 protein at the feto-maternal interface throughout pregnancy in the mare.

A polyclonal human mucin-1 (MUC1) antibody was used to stain immunohistochemically for the presence of MUC1 on the endometrium and fetal membranes in mares between 20 and 309 days of gestation. Western blot analysis demonstrated the presence of a protein equivalent in size to a human MUC1 isoform, MUC1/Y, in equine endometrium, allantochorion and amnion. At all stages of gestation examined immunoreactivity to the MUC1 antibody was detected on the apical surface of the lumenal epithelium of the endometrium and the epithelium lining the mouths and apical regions of the endometrial glands. Furthermore, it persisted unchanged on the surface of the lumenal epithelium lying beneath the highly-invasive chorionic girdle component of the trophoblast before, during and after development of the endometrial cups. The MUC1 immunoreactive protein was also present on the trophoblast and other components of the fetal membranes during the post-fixation, pre-attachment period of gestation (20-40 days) and it persisted on the apical surface of the non-invasive trophoblast of the allantochorion before, during and after attachment, microvillous interdigitation and development of the microcotyledonary epitheliochorial placenta. Hence, the delayed placentation response in mares appears to occur independently of the persistence of an immunoreactive MUC1 protein at the feto-maternal interface.

The Timing of the Maternal Recognition of Pregnancy Is Specific to Individual Mares.

The present experiment aimed at determining whether the timing of the maternal recognition of pregnancy (MRP) was specific to individual mares by determining when luteostasis, a failure to return to oestrus, reliably occurred in individuals following embryo reduction. Singleton (n = 150) and synchronous twin pregnancies (n = 9) were reduced in 10 individuals (5-29 reductions/mare) at pre-determined time points within days 10 (n = 20), 11 (n = 65), 12 (n = 47), 13 (n = 12) or 14 (n = 15) of pregnancy. Prior to embryo reduction, the vesicle diameter was measured in 71% (106/150) of the singleton pregnancies. The interovulatory interval (IOI) was recorded on 78 occasions in seven of the mares in either non-pregnant cycles (n = 37) or those in which luteolysis followed embryo reduction (n = 41). The earliest time post-ovulation at which the embryo reduction resulted in luteostasis in an individual was 252 h (mid-Day 10). Consistency in luteostasis following embryo reduction showed individual variation between mares (272-344 h). Binary logistic regression analysis showed an individual mare effect (p < 0.001) and an effect of the interval post-ovulation at which embryo reduction was undertaken (p < 0.001). However, there was no significant effect of vesicle diameter at the time of embryo reduction (p = 0.099), nor a singleton or twin pregnancy (p = 0.993), on the dependent of luteolysis or luteostasis. The median IOI between individual mares varied significantly (p < 0.05) but was not correlated to the timing of MRP. The timing of MRP varied between the mares but was repeatable in each individual. The factors and mechanisms underlying the individuality in the timing of MRP were not determined and warrant further study.

Lectin histochemistry reveals two cytotrophoblast differentiation pathways during placental development in the feline (Feliscatus).

INTRODUCTION: Placental glycosylation has been examined on eight feline placentae ranging from approximately 15 to 60 days post-conception as little is known about changes in glycan distribution in this species. METHODS: Specimens were resin embedded and lectin histochemistry was applied to semi-thin sections using a panel of 24 lectins and an avidin-biotin revealing system. RESULTS: Abundant tri-tetraantennary complex N-glycan and α-galactosyl residues found in the syncytium in early pregnancy were greatly reduced in mid-pregnancy, though retained at the invasion front in the syncytium (N-glycan) or cytotrophoblast layer (αGal). Some other glycans were also uniquely present in invading cells. Abundant polylactosamine was found in the infolding basal lamina of syncytiotrophoblast and the apical villous cytotrophoblast membrane. Syncytial secretory granules often clustered near the apical membrane abutting maternal vessels. Decidual cells selectively expressed ß-galactosyl residues throughout pregnancy and highly branched N-glycan levels increased over time. DISCUSSION: Glycan distribution changes significantly over pregnancy, probably relating to the development of transport and invasive properties of trophoblast which in the endotheliochorial placenta reaches the level of the maternal vessels. Highly branched complex N-glycans, often associated with invasive cells, N-Acetylgalactosamine and terminal α-galactosyl residues are present at the invasion front abutting the junctional zone of the endometrium. Abundant polylactosamine on the syncytiotrophoblast basal lamina may reflect the presence of specialised adhesive interactions, while clustering of glycosylated granules apically is probably associated with secretion and absorption of material via maternal vasculature. It is suggested that lamellar and invasive cytotrophoblast represent distinct differentiation pathways. 246 words.

Comparative reproduction of the female horse, elephant and rhinoceros: implications for advancing Assisted Reproductive Technologies (ART).

Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we postulate that procedures such as OPU, ICSI, in vitro fertilisation (IVF) and embryo transfer (ET), which are well established in the horse, may represent a basis to develop protocols for endangered pachyderms. In this review, we summarize current knowledge on reproductive physiology relevant to ART. We discuss the current state of ART in all three families and the requirements for the successful implementation of OPU, ICSI, IVF and ET in these species.

Seasonal absence of supplementary corpora lutea in pregnant mares and the relationship with pregnancy loss.

Two experiments were conducted in the Northern (UK) and Southern (Brazil) hemispheres to determine the effect of season (month of conception) on the development of supplementary CL (SCL) and the relationship with pregnancy loss. In experiment 1, 199 pregnancies were followed between Day 14 and term, to determine the number of SCL and pregnancy viability (Northern Hemisphere). From the 199 pregnancies, 178 were obtained from inseminations during the breeding season (March-September), while the rest, 21 pregnancies resulted from conceptions in the non-breeding season (October to February). Pregnancies conceived in the breeding season were more likely (P < 0.01) to have at least 1 SCL (75.8 %, 135/178) than pregnancies from the non-breeding season (33 %, 7/21). However, the pregnancy loss between Days 35 and 120 of pregnancy in mares with no SCL was similar (3.5 %, 2/57; P >0.1) than from mares with SCL (1.4 %, 2/142). In Experiment 2 (Southern Hemisphere), three groups of recipients were used based on their ovarian activity at the time of embryo transfer: Anestrus (n = 8), transitional (n = 7) and cyclic (n = 7) recipient mares. While all transitional and cyclic mares developed at least 1 SCL, only 50 % of anestrous recipients (4/8) developed SCL by 120 of gestation. In conclusion, the development of SCL in pregnant mares is influenced by the time of season of conception, therefore it appears to be regulated by the photoperiod and the endogenous seasonal variation in gonadotropin concentrations. Mares with no SCL were not at increased risk of pregnancy loss.

Bacteria Isolated From Equine Uteri in The United Arab Emirates: A Retrospective Study.

The United Arab Emirates (UAE) presents a unique environment in which to breed horses with a non-physiological breeding season coupled with high temperatures and humidity for much of the year. This study aimed to describe bacterial isolates from the uteri of mares in the UAE and compare them to those reported elsewhere in the world. Bacterial antibiotic resistance was also analyzed to give a starting point for future monitoring. A total of 2,022 swabs taken over five breeding seasons from the endometrium (n = 1,350) or from uterine lavages (n = 672) were submitted for microbiological culture and antibiotic sensitivity testing. At 48 hours post-inoculation 616 of 2,022 (30.5%) of cultures showed microbial growth from which 690 isolates were identified. Most positive plates (548 of 616; 89%) grew one isolate; 68 cultures had two (62 of 616; 10.1%) or three (6 of 616; 1%) isolates. The most frequently isolated bacteria were ß-hemolytic Streptococcus (36.5%; 252 of 690), E. coli (10.6%; 73 of 690), P. aeruginosa (10.1%; 70 of 690), K. pneumoniae (8.8%; 61 of 690) and Aeromonas hydrophila (4.1%; 28 of 690). The lowest level of antibiotic susceptibility for all isolates was shown by trimethoprim-sulphonamide (36.4%; 198 of 544), with amikacin showing the highest (76.1%; 271 of 356). A significant decrease in susceptibility to doxycycline, enrofloxacin, and erythromycin, but a significant increase for amoxicillin with clavulanic acid, was seen for ß-hemolytic Streptococcus. Decreasing susceptibility of trimethoprim-sulphonamide between two time periods was seen for E. coli. Compared to other studies UAE-based mares had a high incidence of P. aeruginosa and K. pneumoniae isolates, whereas E. coli was represented far less frequently as an isolate.

Observations on the glycosylation of the term placenta of the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus): A lectin histochemical study.

INTRODUCTION: Little is known about the glycosylation of placental villi and areolae of cetaceans. Term tissue from the delivered placenta of an Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) was examined using lectin histochemistry to compare trophoblast glycosylation in these two locations. METHODS: Placental blocks fixed in 10% formalin were resin-embedded before semithin sections were stained with 24 biotinylated lectins and an avidin-biotin revealing system. RESULTS: Areolar trophoblast was composed of large, bulbous cells packed with numerous granules compared to the smaller, cuboidal cells clothing the chorionic villi, which had a sparser, mainly subapical granule population. Both were richly glycosylated; generally areolar cells were more heavily stained apart from poor binding to some N-acetylgalactosamine and N-acetylglucosamine termini. Most striking was the distribution of α1,2-linked fucosyl residues, weakly expressed in villous trophoblast but intensely stained in some areolar cells, also terminal sialic acids. Some lectins bound in a variable fashion. Staining of terminal α-d-mannose, which locates mainly to lysosomes, was heavy in areolar cells compared to scattered irregular foci in villous cells. DISCUSSION AND CONCLUSION: The many intracellular inclusions reflect ongoing lysosomal breakdown of histotroph in areolar cells which often show heterogeneous glycosylation staining unlike the uniformly stained villous cells, possibly reflecting partial breakdown of ingested sialoglycoprotein, cell turnover or regional variation in uptake of histotroph. Our results indicate that Dolphin areolae are functionally distinct from villous trophoblast, performing absorptive and phagocytic functions similar to other Artiodactyla.

The effect of embryo reduction and transfer on luteostasis in the mare.

This study investigated the effects of embryo reduction and transfer of Day 11 embryos, with or without subsequent reduction, on luteostasis in the mare. In Experiment 1, reduction of embryos at Days 10 (n = 15), 11 (n = 47), 12 (n = 36), 13 (n = 27), 14 (n = 5) and 16 (n = 2) of pregnancy resulted in luteostasis in 13%, 47%, 78%, 89%, 80% and 100% mares. Mares undergoing > 1 embryo reduction showed consistency in when luteostasis occurred. In Experiment 2, transfer of Day 11 embryos to recipient mares 10 (n = 9), 11 (n = 8), 12 (n = 9) and 13 (n = 8) days post ovulation resulted in luteostasis in 78%, 87.5%, 78% and 37.5% of mares. Only 22%, 37%, 0% and 12%, respectively, of these mares remained pregnant. In the Day 10, 11 and 12 recipients luteostasis occurred on at least one occasion when an embryo was detected at 24 h but not at 48 h post transfer. In the Day 12 recipients luteostasis occurred on three occasions (3/9;33%) when the transferred embryo was not detected at 24 h. In Experiment 3 reduction of a Day 11 embryo 24 h after transfer to a Day 10 (n = 4), 11 (n = 6), 12 (n = 6) or 13 (n = 6) recipient resulted in luteostasis in 100%, 83%, 100%, and 83% of mares. All five Day 11 recipients that had an embryo reduced 12 h post transfer became luteostatic. These results suggest there is plasticity overall, but individual rigidity, in the timing of maternal recognition of pregnancy. Furthermore, an intact embryo need only be present in the uterus for 12 h to cause luteostasis.

The Yolk Sac of the Equine Placenta. Its Remnant and Potential Problems.

This review details the current state of knowledge about the equine yolk sac and its remnant (YSR) in the pregnant mare, which, incidentally, is the only animal species known to exhibit large and/or ossified YSR. It also describes the clinical significance of the YSR and details a case of a strangulating YSR that caused fetal death and abortion.

Successful vitrification of manually punctured equine embryos.

BACKGROUND: 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. OBJECTIVES: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. STUDY DESIGN: In vivo experiment. METHODS: 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 ≤560 µm (n = 10), punctured small (>300 to ≤560 µm; n = 17) and large (>560 µm; 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. RESULTS: Vitrified, manually punctured embryos ≤560 µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos ≤300 µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos >560 µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. MAIN LIMITATIONS: Limited group sizes. CONCLUSION: High pregnancy rates can be achieved by manually puncturing ≤560 µm equine embryos prior to their vitrification and subsequent warming in commercial media.

A new strain of Taylorella asinigenitalis shows differing pathogenicity in mares and Jenny donkeys.

BACKGROUND: Three horse mares inadvertently inseminated with semen from a Tayorella asinigenitalis-positive Jack donkey developed severe, purulent endometritis whereas two Jenny donkeys mated naturally to the same Jack donkey did not develop clinical signs of infection. OBJECTIVES: To isolate and identify the causative agent. STUDY DESIGN: Case report. METHODS: Endometrial swabs from the infected mares were cultured on selective and non-selective media under aerobic and microaerophilic conditions. Isolates were subjected to Gram staining, oxidase and catalase tests, the Monotayl Latex Agglutination test and PCR to test for both T. equigenitalis and T. asinigenitalis. In vitro antimicrobial susceptibility testing was performed and the bacterial isolate was genotyped using MLST. RESULTS: A new sequence type of T. asinigenitalis was confirmed. MAIN LIMITATIONS: A limited numbers of mares and donkeys are described. CONCLUSIONS: This strain of T. asinigenitalis causes a severe venereal infection in mares but not in Jenny donkeys.

Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal-maternal relationship. To this end, we used reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus-mother relationship can be hypothesized.

Successful transfer of day 10 horse embryos: influence of donor-recipient asynchrony on embryo development.

A total of 78 day 10 horse embryos were transferred non-surgically to recipient mares that had ovulated 9, 7, 6, 5, 4, 3, 2 or 1 day after (negative asynchrony), on the same day (synchronous), or 2 or 4 days before (positive asynchrony) the donor (n=6 or 8 mares per group). Pregnancy rates between 100% (6/6) and 63% (5/8) were seen in recipient mares that were between +2 and -6 days asynchronous. Embryo survival to the heartbeat stage declined in recipients that were -7 days asynchronous and no embryos survived in recipients that were -9 days asynchronous. Irrespective of uterine asynchrony, cessation of embryo mobility and fixation at the base of a uterine horn occurred when the conceptus was approximately 17 days old. Conceptus growth and development was slowed when embryos were placed in negatively asynchronous uteri. At the greatest degree of negative asynchrony at which embryos survived to the heartbeat stage, i.e. -7 and -6 days, development of the embryo proper and allantois was retarded. Luteostasis was achieved in recipient mares when day 10 embryos were transferred to recipient mares at any stage of asynchrony between -9 and +2 days with respect to the donor. These results indicate that in the horse, there is a wide window for establishment of pregnancy following embryo transfer to asynchronous recipients. Although progesterone priming of the uterus to a stage equivalent to that of the transferred embryo does not appear to be a prerequisite for embryo survival, it does nonetheless influence embryonic development.

Historical Aspects of Equine Embryo Transfer.

Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, England, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral to the side of ovulation but the rates improved markedly when the whole uterus was flushed on realization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%-90% today. Experimental use of embryo transfer in horses and donkeys demonstrated the unique ability of equids to carry to term a full range of interspecies hybrid conceptuses and extraspecies pregnancies created by embryo transfer. Furthermore, splitting of day 4-8 cell embryos and day 6 compact morulae allowed the creation of genetically identical twin foals. But despite these and other significant advances over the past 45 years, a persisting limitation is the relatively low embryo recovery rates from donor mares treated with exogenous gonadotropins in attempts to induce them to superovulate. This is due to the toughness of the ovarian tunica albuginea which forces ovulation through the ventrally situated ovulation fossa where multiple follicles compete with each other and luteinize before they can ovulate properly.

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.