Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation.

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na+/K+ gradient, which is dependent on an Na+-K+ antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

Expression of leukaemia inhibitory factor at the conceptus?maternal interface during preimplantation development and in the endometrium during the oestrous cycle in the mare.

Leukaemia inhibitory factor (LIF) plays a critical role in blastocyst development and implantation in several species. The present study investigated mRNA and protein expression for LIF, as well as the low-affinity LIF receptor (LIFR) and interleukin-6 signal transducer (IL6ST), in equine endometrium, trophoblast and histotroph during early pregnancy and in the endometrium during the oestrous cycle. Endometrial LIF mRNA expression was upregulated after Day 21 of pregnancy, whereas LIF immunoreactivity increased in the endometrium on Day 28. Expression of LIF mRNA in the yolk sac membrane increased from Day 21 of pregnancy, whereas LIF immunoreactivity increased from Day 28 in the trophoblast. LIFR and IL6ST mRNA was expressed in the endometrium during both the oestrous cycle and early pregnancy and, although LIFR and IL6ST protein were localised to the glandular epithelium during the cycle and first 14 days of pregnancy, from Day 21 they were located in the luminal epithelium. Trophoblast expression of LIFR and IL6ST increased as pregnancy proceeded. In conclusion, LIF expression increased at the conceptus-maternal interface during capsule attenuation. Because contemporaneous upregulation of both LIFR and IL6ST was also observed in the trophoblast, we propose that LIF plays an important role in the development of endometrial receptivity for trophoblast growth, apposition and adhesion in mares.

Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare.

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.

Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen.

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p<0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p<0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Phenotypic characteristics of hydrocephalus in stillborn Friesian foals.

Hydrocephalus is uncommon in horses. However, in recent years, it has become clear that the prevalence of hydrocephalus is greater in Friesian horses than in other breeds probably due to their limited gene pool. Before identification of candidate genes that predispose to the development of hydrocephalus in Friesian horses can be pursued, an in-depth, phenotypic, pathological description of the condition in Friesians would be of great benefit. Our study aimed to characterize the morphology of hydrocephalus in Friesian horses, to support further investigation of the genetic background of this condition. Four stillborn Friesian foals with hydrocephalus were examined macroscopically and microscopically and compared with 2 normal stillborn Friesian foals without hydrocephalus. In all clinical cases, tetraventricular and venous dilatations were observed, together with malformation of the petrosal bone and, as a result, narrowing of the jugular foramen. These observations suggest a communicative hydrocephalus with a diminished absorption of cerebrospinal fluid into the systemic circulation at the venous sinuses due to a distorted, nonfunctional jugular foramen. This type of hydrocephalus is also recognized in humans and dogs and has been linked genetically to chondrodysplasia; this has already been recognized in dwarfism, which is another monogenetic defect in Friesian horses.

Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing.

The cryotolerance of equine blastocysts larger than 300 µm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 µm, n = 14 and > 550 µm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300-550 µm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 µm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.

Effect of cooled storage on quality and DNA integrity of Asian elephant (Elephas maximus) spermatozoa.

Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI.

Cryopreservation of equine embryos: current state-of-the-art.

During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the following: (i) the absence of commercially available products for reliably stimulating superovulation; (ii) very poor pregnancy rates following cryopreservation of embryos >300 µm in diameter; (iii) difficulty in recovering embryos at early developmental stages amenable to cryopreservation; and (iv) interembryo variation in susceptibility to cryodamage. However, acceptable success rates (>55% pregnancy) have been reported for both slow-frozen and vitrified small embryos (<300 µm), and there is renewed interest in cryopreservation, not only in the context of standard ET programmes, but also because it would facilitate pre-implantation genetic testing and allow wider access to techniques for producing embryos in vitro, such as intracytoplasmic sperm injection and nuclear transfer. This article will review the current status of equine embryo cryopreservation.

Success rate in a clinical equine in vitro embryo production program.

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0-13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating.

Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos.

BACKGROUND: Monozygotic multiple pregnancy is rare in horses, but may be more common after transfer of an in vitro produced (IVP) embryo. OBJECTIVES: To determine the occurrence, incidence, characteristics and outcome of monozygotic siblings arising from in vivo and IVP equine embryos. STUDY DESIGN: Retrospective case series. METHODS: A total of 496 fresh in vivo and 410 frozen-thawed IVP blastocysts, produced by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes from Warmblood mares, were transferred into recipient mares. The likelihoods of pregnancy and multiple pregnancy were calculated, and the clinical features and outcome of any multiple pregnancy were recorded. RESULTS: The likelihood of pregnancy after transfer of a single IVP or in vivo embryo was 62% (254/410) and 83% (413/496) respectively. The incidence of multiple pregnancy was 1.6% (4/254) and 0% (0/413) for IVP and in vivo blastocysts, respectively. More specifically, three IVP blastocysts yielded twin embryo propers/fetuses, and one IVP conceptus developed three distinct embryonic bodies. Interestingly, only one embryonic vesicle was detected at all ultrasonographic examinations prior to embryo proper development. Multiple embryonic bodies only became apparent at later scans to check for an embryo proper and heartbeat, or when the recipient mare aborted. Two twin pregnancies aborted spontaneously at 3 and 9 months, respectively, while the heartbeat was lost from all three embryos in the triplet pregnancy before day 35 of gestation. Twin reduction by per rectum compression of one fetal thorax was attempted at day 50 of gestation in the fourth case; however, both fetuses were lost. MAIN LIMITATIONS: Small number of cases. CONCLUSIONS: In vitro embryo production resulted in a higher incidence of multiple monozygotic pregnancy, which could only be diagnosed after development of the embryo proper and is likely to result in pregnancy loss later in gestation if left untreated.

Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes.

Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (approximately 30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05).

Normal function of the hypothalamic-pituitary growth axis in three dwarf Friesian foals.

Serial blood samples were collected from three dwarf Friesian foals to examine their endogenous growth hormone (GH) profiles, and the integrity of the GH-insulin-like growth factor-1 (IGF-1) axis was tested in one of them by examining its responses to the administration of GH-releasing hormone (GHRH) and to 10 days of treatment with recombinant equine GH. The basal serum concentrations of IGF-1 in the three dwarf foals were compared with those in nine age-matched normal foals. All the dwarf foals secreted endogenous GH. Stimulation with 7.0 microg/kg GHRH led to a 1400 per cent increase in plasma GH concentration in the dwarf foal tested, and 10 daily subcutaneous treatments with 20 microg/kg recombinant equine GH led to a 100 per cent increase in its serum IGF-1 concentration. The basal serum concentrations of IGF-1 in the dwarf foals were not significantly different from those of the normal foals.

Small day 8 equine embryos cannot be rescued by a less advanced recipient mare uterus.

Equine embryos tolerate an unusually large degree of negative uterine asynchrony (recipient mare up to 5 days behind the donor mare). By contrast, positive asynchrony of more than 2 days results in a high incidence of early embryonic loss (EEL). Day 8 embryos range in diameter from approximately 130-1300 µm, with embryos smaller than 300 µm reported to suffer an increased incidence of EEL. However, it is not known whether this reduced viability is due to intrinsically poor embryo quality, or to inadvertent recipient uterine stage-embryo (positive) asynchrony. To examine whether small embryos survive better in Day 4-5 recipients than in recipients with a more advanced uterine stage, the likelihood of pregnancy (PR) and EEL for 62 small (<300 µm) and 215 larger Day 8 horse embryos were compared after transfer to recipients at different uterine stages (Days 4-5, 6-7 and 8-9) using logistic regression. Overall, EEL was higher (21.2%; P < 0.05) for small than larger embryos (7.1%). However, neither PR nor EEL were influenced by the recipient's uterine stage at the time of transfer (P > 0.1). The EEL for small embryos transferred into Day 4-5, 6-7 and 8-9 recipients was 20.8, 18.7 and 25.0%, respectively. We conclude that embryos recovered on Day 8 with a diameter <300 µm are at increased risk of EEL due to reasons other than inadvertent positive asynchrony with the recipient mare's uterus.

Advanced mare age impairs the ability of in vitro-matured oocytes to correctly align chromosomes on the metaphase plate.

BACKGROUND: Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. OBJECTIVES: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro-matured (IVM) oocytes. STUDY DESIGN: Morphometric and morphological analysis. METHODS: To investigate differences in spindle organisation and chromosome alignment between young and old mares, oocytes collected from slaughtered mares were divided into two groups depending on mare age (young, ≤14 years and old, ≥16 years), IVM and stained to visualise chromatin and alpha-tubulin. Spindle morphology, morphometry and chromosome (mis)alignment were evaluated by confocal microscopy and 3D image analysis. RESULTS: Oocytes from old mares showed a higher incidence of chromosome misalignment (47.4% vs. 4.5%; P<0.001) and a thicker metaphase plate (mean ± s.d.: 5.8 ± 1.0 µm vs. 4.9 ± 0.9 µm; P = 0.04) than oocytes from young mares. Although no differences in spindle morphometry were detected between old and young mares, an increased major spindle axis length was associated with chromosome misalignment (mean ± s.d.: 25.3 ± 6.1 µm vs. 20.8 ± 3.3 µm; P = 0.01) irrespective of age. MAIN LIMITATIONS: The oocytes were IVM and may not exactly reflect chromosome misalignment in vivo. CONCLUSIONS: Advanced mare age predisposes to chromosome misalignment on the metaphase II spindle of IVM oocytes. The compromised ability to correctly align chromosomes presumably predisposes to aneuploidy in resulting embryos and thereby contributes to the age-related decline in fertility and increased incidence of early pregnancy loss. The Summary is available in Portuguese - see Supporting Information.

The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm.

The epididymis is a long, tightly coiled tube within the lumen of which sperm matures. Sperm maturation involves morphological and biochemical changes in the sperm plasma membrane in response to epididymal secretions and their various proteins. Some of these proteins become outer membrane components while others become integral membrane proteins; transfer of some proteins to the sperm plasma membrane may be mediated by epididymosomes. Nevertheless, the molecular pathways by which spermatozoa acquire fertilizing capacity during their transit through the epididymis remain ambiguous. In a recent study of stallion epididymal sperm, we found that sperm harvested from different parts of the epididymis (caput, corpus and cauda) had a varying, but generally poor, ability to undergo the acrosome reaction in vitro. At ejaculation, however, sperm mix with seminal plasma which contains various components, including the small membranous vesicles known as prostasomes, that may enable the sperm to undergo physiological activation. Seminal plasma components may have a 'washing' effect and help to remove 'de-capacitation' factors that coat the sperm during storage in the cauda epididymis; alternatively seminal plasma and prostasomes may contain factors that more directly promote sperm activation. This article reviews current information on the roles of epididymal and accessory gland fluids on the acquisition of fertilizing capacity by stallion sperm.

Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses.

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.

Phenotypic diagnosis of dwarfism in six Friesian horses.

An extreme form of abnormal development, dwarfism, is common in man and some animals, but has not been officially reported in horses. Within the Friesian horse breed, congenital dwarfism has been recognised for many years, but no detailed report exists on its phenotype. The most salient feature of the dwarf syndrome is the physeal growth retardation in both limbs and ribs. Affected animals have approximately 25% shorter fore- and hindlimbs and approximately 50% reduced bodyweight. Postnatal growth is still possible in these animals, albeit at a slower rate: the head and back grow faster than the limbs and ribs leading to the characteristic disproportional growth disturbance. Thus, adult dwarfs exhibit a normal, but a relatively larger head conformation, a broader chest with narrowing at the costochondral junction, a disproportionally long back, abnormally short limbs, hyperextension of the fetlocks and narrow long-toed hooves. Furthermore, a dysplastic metaphysis of the distal metacarpus and metatarsus is radiographically evident. Microscopic analysis of the growth plates at the costochondral junction shows an irregular transition from cartilage to bone, and thickening and disturbed formation of chondrocyte columns, which is similar to findings in osteochondrodysplasia.

Effects of exogenous insulin on luteolysis and reproductive cyclicity in the mare.

Insulin is a pancreatic hormone that classically regulates carbohydrate and fat metabolism, but also appears to play a role in various reproductive processes. A preliminary study suggested insulin production by day 10 to 18 equine conceptuses. The aim of the present study was to examine the hypothesis that insulin is the conceptus signal responsible for maternal recognition of pregnancy (MRP) in the mare, or otherwise influences reproductive cyclicity during the MRP period. Six Warmblood mares were treated daily during days 7 to 17 after ovulation of two successive oestrous cycles with either (short and intermediate acting) insulin or control saline. Mares were assigned randomly to treatment, and crossed over during the subsequent cycle. Time of ovulation and corpus luteum surface area were determined by serial transrectal ultrasonographic examination of the mares' ovaries, and daily jugular vein blood samples were analysed for progesterone and luteinizing hormone (LH) concentrations. On day 14 of dioestrus, the luteolytic drive was examined by measuring systemic 15-ketodihydroprostaglandin F(2 alpha) (PG-metabolite) release in response to oxytocin challenge. In addition, yolk sac fluid recovered from 32 day 10 to 14 equine conceptuses was analysed for insulin concentrations. Insulin administration did not affect luteal size, dioestrus length, the interovulatory interval, or circulating LH concentrations. Insulin administration also failed to suppress oxytocin-induced PGF(2 alpha) release, and tended to depress systemic progesterone concentrations. Finally, insulin could not be detected in the yolk sac fluid of day 10 to 14 equine conceptuses by radio-immunoassay. It is concluded that insulin administered daily during days 7 to 17 of dioestrus has little or no effect on reproductive cyclicity in the mare, and is unlikely to be the MRP signal.

Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares.

The diameter of embryos recovered from mares on Day 8 after ovulation varies greatly, from as little as 130 µm to as much as 2500 µm. Several factors have been proposed to affect embryo size at recovery, one of which is the type of semen (frozen vs fresh or cooled-transported) used to inseminate the mare. In addition, it has been shown that smaller embryos (<300 µm) recovered on Day 8 are less likely than larger embryos to result in successful pregnancy after transfer. However, whether the actual age of the embryo (interval from fertilization to flushing) in relation to its size also influences the post-transfer viability is unclear. The aims of this study were: a) To determine the effect of semen type (frozen-thawed vs cooled-transported) on embryo diameter after pre-ovulatory insemination; and b) To establish the relationship between embryonic age, embryo size and likelihood of pregnancy and pregnancy loss following transfer. A total of 179 embryos were recovered from mares inseminated with: frozen semen post-ovulation 8 days previously (G1; n = 35); cooled-transported semen pre-ovulation 8.5 days previously (G2; n = 95); frozen semen pre-ovulation 8.5 days previously (G3; n = 30); and frozen semen post-ovulation 9 days previously (G4; n = 19). The effect of embryonic age, type of semen, donor mare and its age, number of ovulations and embryos per flush on embryo diameter was tested using a general linear model of variance. In addition, the proportions and survivals of small embryos (<300 µm) in each group were compared with those of respective larger embryos by Fisher's exact test. Embryonic age (P < 0.001) and age of the donor mare (P = 0.07), but no other factor, influenced embryo diameter. The proportion of small embryos was 42.9, 10.5, 10.0 and 10.5% for Groups 1, 2, 3 and 4, respectively. The pregnancy status of recipient mares 35 days post-transfer for small embryos from Group 1 (12/15; 80.0%) was not different (P > 0.1) from that of recipients of small embryos from Groups 2 to 4 combined (8/15; 53.3%).

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years.

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.