Haflinger Mares Research Articles

Congenital hydrocephalus in a stillborn Haflinger foal

A 14-year, multiparous Haflinger mare, apparently fullterm (unknown breeding or ovulation date), was presented for dystocia. Mare was transvaginally palpated at the farm by the referring veterinarian; foal was in craniolongitudinal presentation, dorsosacral position with extended forelimbs in the vaginal vault and head just cranial to cervix, and a cranial abnormality of the foal prevented assisted vaginal delivery. Mare was referred. Foal was not alive at presentation and was undeliverable with assistance because of congenital hydrocephalus. Anesthetized mare was placed in Trendelenburg position, controlled vaginal delivery was elected and foal was removed via fetotomy. Mare was negative for β1, 3- N-acetylgalactosaminyltransferase 2 (B3GALNT2) nonsense mutation (reported to be responsible for hydrocephalus). Dystocia in a Haflinger mare because of hydrocephalus is reported for the first time.

Estimation of the genetic parameters for temperament in Haflinger horses based on the results of a character test using a pedigree-based relationship matrix

Literature provides indications that the personality and temperament of a horse are sufficiently genetically determined. This background was the impetus to continue collecting phenotypic information on the temperament in the South Tyrolean Haflinger horses, not only to have marketing tools but also to be able to estimate genetic parameters for a future genetic evaluation. The latter should open the possibility to include temperament traits in the breeding goal. Therefore, the aim of the present study was to consider for the very first-time phenotypic information from character testing for estimating genetic parameters for temperament in Haflinger horses. For the present study, data from routine character testing of 210 South Tyrolean Haflinger mares between 2019 and 2021 were considered. A multivariate estimation of heritability (h2) and genetic correlations (r) was carried out for the criteria ‘Interest’, ‘Activity’ and ‘Excitement’. In addition, univariate and bivariate estimation runs were also carried out. The pedigree-based heritability for the behavioural trait Interest was lowest with 0.07 and a standard error of ±0.04. For the other two criteria, Activity and Excitement, h2 was 0.19 ± 0.05 and 0.21 ± 0.04, respectively. The genetic correlation between Interest and Activity and between Interest and Excitement was in both cases negative with −0.52 and −0.61, respectively while the genetic correlation between Excitement and Activity was positive (0.99). Results of the present study highlight the potential of considering temperament traits in breeding of Haflinger horses.

Possible involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares

In horses, resumption of ovarian function after parturition occurs early and the majority of mares ovulate within two weeks after parturition. Previous research suggests that early postpartum increases in the peripheral concentration of prolactin and somatotrophic hormones are the major stimulus for resumption of ovarian activity and ovulation. In the present study, we investigated if placental tissue may be a source of somatotrophic hormones and thus influence ovarian activity. Ovarian follicular development and concentrations of prolactin, insulin like growth factor-1 (IGF1) and LH were determined in 14 healthy Haflinger mares (9.2±3.8 years). From D15 before until D15 after parturition (pp), transrectal ultrasonography of ovarian activity was performed and blood samples were collected for determination of hormone concentrations (prolactin, IGF1 by ELISA; LH by RIA). All foals were healthy, mature and stayed with their dams. Samples from the allantochorion and amnion were obtained immediately after delivery. The relative abundance of mRNA for IGF1 and the prolactin receptor (PRLR) was analyzed by qPCR. Formalin fixed samples were analyzed for protein expression of IGF1 and PRLR by immunohistochemistry. Statistical analysis (IBM SPSS 27.0) was done by analysis of variance (for repeated measures) comparing data from mares that ovulated(group ov; n=5) and did not ovulate until D15 pp (group non-ov, n=9). Values are mean ± SD. Mares from group ov foaled later in the year than non-ov mares (day of year 84±29 vs. 50±20, p<0.05), but no differences in gestation length, placental weight or size were detected. Already on D15 before parturition, ovarian follicles <2 cm diameter were present in all mares, but growth to preovulatory size occurred only pp with a higher growth rate in ov mares (day x group p<0.05). Prolactin and IGF1 concentrations increased from D5 before parturition, peaked at D1 pp (ov vs. non-ov: prolactin 13.0±6.3 vs. 7.9±4.4 ng/ml, p<0.05; IGF1 409±71 vs. 350±65 ng/ml, p<0.05) and declined thereafter. The LH concentration increased from D1 pp in group ov (D12 pp: 23.82±15.96 ng/ml) but not in non-ov (D12 pp: 10.82±5.26 ng/ml; day x group p<0.001). There was a high abundance of mRNA and protein for IGF1 and PRLR in placental tissue irrespective of group. In conclusion, ovarian follicles are detectable before foaling, but develop to preovulatory size only pp when prolactin and LH concentrations increase. In agreement with the situation in cattle, IGF1 stimulates ovarian activity in lactating mares. Placental tissue produces IGF1 that may be under the influence of prolactin as suggested by the presence of PRLR in both amnion and allantochorion.

Increased AMH plasma concentration and small antral follicles - Deslorelin slow release implants as a promising tool for preparation of mares for OPU?

A major factor affecting oocyte yield during ovum pick-up (OPU) procedures in mares is the number of follicles present on the ovary and the associated number of recovered oocytes (Cuervo-Arango et al. Reproduction, Fertility and Development. 2019; 31(12): 1894-1903). The interest in manipulation of ovarian follicular populations in horses is increasing because of benefits with regard to the oocyte yield and the overall outcome for assisted reproductive procedures. This study aimed to evaluate effects of slow-release implants containing deslorelin acetate on antral follicle counts (AFC), follicular subpopulations of different size ranges and plasma anti-Mullerian hormone (AMH) and gonadotrophin concentration in mares. Haflinger mares (n=12) were synchronized with two injections of a PGF2α analog. One day after the second injection, mares were randomly assigned to the slow-release implant (DES, 9.4mg deslorelin implant, n=6) or the control group (CON, deslorelin short-acting formulation, 1.25mg, n=6). Transrectal examination of the genital tract and collection of blood samples for the analysis of progesterone, AMH and gonadotrophins were performed until day 90 and on days of AFC assessment. AFC and follicle numbers according to size groups were determined on days 0, 10, 25, 50, 90, 180, 270 and 360 after implant insertion. The interval from implant insertion to the first spontaneous ovulation was prolonged in group DES (23.8±10.5 days) compared to CON(17.0±3.9 days) (p<0.05). Treatment influenced concentrations of LH and FSH over time (p<0.05) with the concentration of LH being greater in DES than in CON mares on days 30 and 55 and of FSH on day 15 after implant insertion, respectively (p<0.05). AMH concentration was greater in group DES from day 30 onwards (interaction treatment x time p<0.05). The AFC and the number of follicles with 5 to 10, 10 to 15 and 15 to 20 mm in diameter changed over time (p<0.05). A time x treatment interaction was demonstrated for follicles of 10 to 15 mm in diameter with more follicles of this size present in DES than in CON mares on day 90 (p<0.05). In group DES, concentration of AMH and the AFC (r=0.623, p<0.001) as well as concentration of AMH and the number of follicles 5 to 20 mm in diameter (r=0.442, p<0.01) were positively correlated whereas there was no such correlation in group CON. In conclusion, long-acting deslorelin implants induce changes in follicular development reflected in AMH plasma concentration. These changes are present from day 30 after implant insertion, suggesting that not only direct effects of deslorelin on gonadotrophin secretion but also the reduction of FSH suppressing factors from follicles such as inhibin may be involved. Treatment of mares with 9.4mg deslorelin slow-release implants might thus be a promising tool as preparation for OPU.

Продължителност на еструса и половия цикъл при кобили от породата Хафлингер

A study of the duration of oestrus and estrous cycles in mares from Haflinger breed (HB) and the influence of some factors was conducted. The records used for this study were from the breeding registers of the mares from the State Stud farm Kabiuk near Shumen, covering a period of 11 years (2009 to 2020). In the study were included 15 mares with 110 estruses and 22 estrous cycles. With the mares from Haflinger breed, the average duration of oestrus is 5.52 ± 0.26 days and the length of the estrous cycle is 24.21 ± 1.13 days. The year (P<0.001), month (P<0.001), season (P<0.05), and length of the estrous cycle - the year of fertilization (P<0.05) had reliable influence on the duration of oestrus. In Haflinger mares, the shortest estrus is in the middle and at the end of the breeding season (March: 4.39 ± 0.46 days, July: 3.33 ± 0.82 days). Its duration increases during the winter season and reaches its peak in the middle of spring (February: 5.96 ± 0.51 days, April: 8.37 ± 0.72 days).

Deslorelin Slow-Release Implants Delay Ovulation and Increase Plasma AMH Concentration and Small Antral Follicles in Haflinger Mares.

Simple SummaryIn horses, oocyte collection followed by intra-cytoplasmatic sperm injection is increasingly used. The yield of oocytes is a limiting factor and depends on the number of follicles present on the ovary during oocyte collection. Therefore, the aim of this study was to analyze the effect of slow-release implants containing the GnRH analogue deslorelin on the number of follicles and on hormones regulating follicular development. Six mares received a deslorelin implant and six mares served as controls. The interval to the first spontaneous ovulation was prolonged in treated mares. The treatment changed the release pattern of the gonadotrophins LH and FSH. Changes in the number of follicles 10 to 15 mm in diameter were detected in deslorelin-treated mares. These changes were also reflected by increasing plasma anti-Muellerian hormone concentrations, a hormone produced by growing follicles. In conclusion, deslorelin implants induce changes in ovarian follicle subpopulations and could be a promising tool for the preparation of mares for assisted reproductive procedures.There is an increasing interest in the manipulation of ovarian follicular populations in large domestic animals because this could prove beneficial for assisted reproductive techniques such as ovum pick-up (OPU). The aim of the present study was to evaluate the effects of deslorelin slow-release implants (SRI) on the interovulatory interval, antral follicle count (AFC), number of follicles of different size ranges and plasma anti-Muellerian hormone (AMH) concentration in mares. To synchronize their estrous cycles, Haflinger mares (n = 12) were treated twice with a PGF2α analogue. One day after the second injection (day 0), mares received a 9.4 mg deslorelin SRI (group DES, n = 6) or 1.25 mg deslorelin in a short-acting formulation (CON; n = 6), respectively. Regular transrectal ultrasonography of the genital tract was performed and blood samples were collected for the analysis of progesterone, AMH and gonadotrophins. The interval from implant insertion to the first spontaneous ovulation was 23.8 ± 10.5 days in group DES compared to 17.0 ± 3.9 days in group CON (p < 0.05). For the concentrations of LH, FSH and AMH, interactions between time and treatment were detected (p < 0.05). The AFC and the mean number of follicles with 5 to 10, 10 to 15 and 15 to 20 mm in diameter changed over time (p < 0.05). A time x treatment interaction was demonstrated for follicles of 10 to 15 mm in diameter (p < 0.05). The changes in this follicular subpopulation were reflected by increased plasma AMH concentration in group DES. In conclusion, 9.4 mg deslorelin implants show minor effects with regard to estrus suppression in mares, whereas the changes in the subpopulation of small ovarian follicles could be a promising tool for preparation of mares for OPU.

Transport-related stress in five-day-old foals and their dams

Horses are frequently transported by road but little is known about the stress response of transported newborn foals. In this study, we have analyzed the stress response of postpartum Haflinger mares (n = 6 over two years) and their 5-day-old foals (n = 12) to a 20 km road transport in a trailer with the middle partition either removed or separating the mare and her foal. Noninvasive techniques were used to analyze the physiological stress variables such as salivary cortisol, heart rate, and heart rate variability (HRV). Mares and their foals responded to transport with an increase in cortisol concentration (P < 0.001). The increase was more pronounced and prolonged in foals compared to their mothers (P = 0.001). Heart rate increased in mares and foals during transport (P < 0.001) but this increase was less pronounced in foals compared to mares (P = 0.001). The HRV increased during transport (P < 0.05) in mares but remained constant in transported foals. The presence or absence of the partition was largely without effect on the physiological stress variables determined. In conclusion, 5-day-old foals respond to road transport with a clear adrenocortical activation which was more pronounced than in their dams. There were no changes of HRV in transported foals.

61 Low plasma progestin concentration during the early luteal phase impairs equine conceptus development until placentation

During the early luteal phase, low progesterone concentrations delay downregulation of endometrial progesterone receptors. This contributes to impaired histotroph production at Day 14 of pregnancy (Beyer et al. 2019 Theriogenology 125, 236-241). Until the beginning of placentation (i.e. Day 37 of pregnancy), nutritional supply of the equine conceptus depends on histotrophy alone. The aim of the present study was to analyse development of the equine conceptus under reduced plasma progesterone concentrations until shortly before placentation. Fertile Haflinger mares (n=11; 4-11 years old) were examined daily by transrectal ultrasonography, and when in oestrus, they were inseminated every 48h until spontaneous ovulation. Mares were randomly assigned to either the treatment group and received the prostaglandin F2α agonist cloprostenol (PGF2α; 125μg) once daily for 3 days after ovulation (Beyer et al. 2019) or the control group and left untreated. After conceptus collection on Day 34, mares were allowed one oestrous cycle for recovery and subsequently assigned to the opposite treatment, thus serving as their own controls. From Day 10 after ovulation, conceptus development including size, uterine fixation, and heartbeat detection was evaluated daily by transrectal ultrasonography. On Day 34, conceptus and fetal membranes were recovered transcervically. The recovered material was weighed, measured, and stored for further analysis. Conceptuses underwent microcomputed tomography that was evaluated by Amira (Thermo Fisher Scientific). Statistical comparison for differences between control and treatment pregnancies was performed by nonparametric Wilcoxon test or chi-square analysis. The day of first visualisation of the conceptus and of the embryo proper and heartbeat did not differ between treatments. Uterine fixation occurred on Day 19.3±0.5 in conceptuses from PGF2α-treated pregnancies but on Day 16.7±0.4 in controls (P&amp;lt;0.05). At 34 days of gestation, the conceptuses from PGF2α-treated mares were smaller (P&amp;lt;0.05) than control conceptuses when measured with ultrasound callipers in utero (maximal length: PGF2α 17.4±0.3mm, control 19.0±0.4mm). Conceptus weight determined after recovery was less in treated pregnancies (PGF2α 2.5±0.2 g; control 3.5±0.3 g; P&amp;lt;0.05). Microcomputed tomography analysis of selected inner organs showed some differences in development. Seven complete pairs of undestroyed conceptuses were available. Mean numbers of 33.4±3.2 and 18.8±7.5 bronchi were detected in control and PGF2α conceptuses, respectively (P&amp;lt;0.05). Heart volume did not differ, but communication between ventricles was detected in only 1/7 control but 5/7 PGF2α conceptuses (P&amp;lt;0.05). The footpad was present in 6/7 control and 0/7 PGF2α conceptuses (P&amp;lt;0.001). In conclusion, subphysiological progesterone concentration during the early luteal phase delays development of the equine conceptus before placentation. The condition may contribute to early conceptus loss in horses, which occurs in up to 20% of pregnancies.

Strangulation of the duodenum just oral to its caudal flexura caused by a volvulus

SummaryAn 8‐year‐old Haflinger mare with a 10‐day‐old foal at foot was referred with mild abdominal pain. On the basis of clinical examination, blood and peritoneal fluid analysis, the cause of pain was suspected septic peritonitis. The horse was initially managed medically, with the horse placed in box confinement under close monitoring. No permission was given for an exploratory laparotomy, and it was decided to perform a diagnostic laparoscopy in the standing horse. The laparoscopy revealed a 360‐degree anticlockwise partial duodenal volvulus of the caudal descending duodenum, which was manually reduced (hand‐assisted laparoscopy). After the release, the mesentery of the duodenum showed an ischaemic band (yellowish white and dark red with a hyperaemic side) and the duodenum had a distinct ischaemic ring (orally and aborally) distinguishing the incarcerated part from the normal duodenum. Ten minutes after the release, the colour remained the same. In conjunction with the owner, it was decided to euthanise the pony. Pathology confirmed the presence of a strangulation ring and the ischaemic condition of the involved duodenum.

Long term consistency and location specificity of equine gluteus medius muscle activity during locomotion on the treadmill

BackgroundThe equine m. gluteus medius (GM) is the largest muscle of the horse, its main movement function is the extension of the hip joint. The objective of the present study was to measure equine GM activity in three adjacent locations on GM during walk and trot on a treadmill, in order to document potential differences. Fourteen Haflinger mares were measured using surface electromyography and kinematic markers to identify the motion cycles on three occasions over 16 weeks. The electrodes were placed on left and right gluteus medius muscle over the middle of its widest part and 5 cm lateral and medial of it. For data processing, electrical activity was normalised to its maximum value and timing was normalised to the motion cycle. A Gaussian distribution approach was used to determine up to 10 modes of focussed activity, and results were analysed separately for stance and swing phase of the ipsilateral hindlimb.ResultsFair reliability was found for mean mode values (Cronbach’s alpha = 0.66) and good reliability was found for mean mode locations (Cronbach’s alpha = 0.71) over the three data collection days. The magnitude of muscle activity identified as mean mode value was much larger at trot than at walk, and mean mode value was significantly different between stance phases of walk and trot for all electrode positions (p < 0.01). The pattern of muscle activity identified as mean mode location was significantly different for walk and trot at all electrode positions, both during stance and swing phases (p < 0.001). This indicates the different timing pattern between the gaits. Results of the three electrode positions on the same muscle during each gait were not significantly different when comparing the same measurement.ConclusionsThe middle of the equine GM does not show any indication of functional differentiation during walk and trot on a treadmill; this might be due to lack of segmentation as such, or due to lack of need for segmented use for these very basic main tasks of the muscle. The reliability of the sEMG measurements over several weeks was fair to good, an indication for the robustness of the methodology.

Effect of Diet on Haflinger Horses With GYS1 Mutation (Polysaccharide Storage Myopathy Type 1)

Describing the effects of a carbohydrate-rich diet (CRD) on Haflingers with R309 H glycogensynthase 1 (GYS1) mutation. Haflinger mares heterozygous positive (polysaccharide storage myopathy [PSSM] type 1, n = 7) and negative (control, n = 7) for GYS1 mutation received 6 weeks of CRD and hay only (hay), with a wash out period of two. Each diet was followed by oral glucose tolerance test (oGTT), submaximal treadmill exercise test (STET), and biopsies of semitendinosus (STm) and gluteus medius (GMm) muscles. Insulin sensitivity was evaluated by oGTT. Plasma creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and lactate were measured before and after STET. Semitendinosus and GMm biopsies were evaluated for distribution of fiber types (1, 2A, and 2X), chronic myopathic changes, and complex Periodic Acid Schiff (cPAS) positive amylase resistant inclusions. One Haflinger of control group had cPAS inclusions indicating PSSM type 2; therefore, it was excluded. In PSSM1 horses, cPAS were found in every STm sample, and in 10 of 14 GMm samples, furthermore, STm samples showed a greater total myopathy score and significantly more type 2A and fewer type 2X fibers compared with controls. There were no significant differences between groups regarding enzyme levels and for oGTT. Diet had no effect on muscle enzyme levels after STET and on cPAS inclusions in PSSM1 group. Glycogensynthase 1 mutation in Haflingers was not accompanied by clinical signs, even after STET. Carbohydrate-rich diet for 6 weeks did not induce clinical signs or increase cPAS inclusions. As PSSM type 2 is a possibility in Haflingers, STm muscle biopsy and cPAS evaluation should be performed if GYS1 gene test is negative.

Abdominal Wall Mass and Hemoabdomen in a Haflinger Mare

A 6-year-old Haflinger mare was presented with a history of recurrent hemoabdomen. On necropsy, a firm infiltrative multinodular yellow mass was observed in the wall of the posterior abdomen. Histopathologic examination revealed a proliferation of fibroblastic cells, which were positive for α-smooth muscle actin and vimentin.

Disseminated lipid-rich peritoneal mesothelioma in a horse

A 9-year-old Haflinger mare presented to the Liphook Equine Hospital with a history of weight loss, azotemia, and repeated episodes of ascites over a period of 10 days. The horse was euthanized after exploratory laparotomy revealed large numbers of variably sized masses distributed throughout the peritoneal cavity. Macroscopically, some masses were papillary, while others were nodular. Histologically, the masses were comprised of large to giant, variably shaped, and occasionally multinucleated neoplastic cells with marked anisokaryosis and anisocytosis and a high mitotic rate. Small to moderate numbers of neoplastic cells were swollen by 1 to several, moderately sized to large, clear, circular or ovoid vacuoles, which stained positive with oil red O. Immunohistochemically, the neoplastic cells co-expressed vimentin and cytokeratin. Electron microscopy demonstrated tumor cells with tight junctions, microvilli, and numerous intracytoplasmic lipid droplets. These findings are consistent with a lipid-rich form of mesothelioma, which should be considered as a differential diagnosis if lipid vacuoles are present in potentially neoplastic cells in equine abdominocentesis samples.

Ultrasonographic Morphometric Measurements of Digital Flexor Tendons and Ligaments of the Palmar Metacarpal Region in Haflinger Horses

The aim of the study was to determine morphometric values of the superficial and deep digital flexor tendon, the inferior check ligament and the suspensory ligament in the palmar metacarpal region of sound Haflinger horses using ultrasonography and to assess the influence of different factors on them. The findings were compared with the literature. A total of 30 sound Haflinger mares aged 4-20 years were used. Transverse ultrasound images were obtained with an 8-10 linear MHz transducer and the cross-sectional area, the circumference and the dorsopalmar and lateromedial width were determined for each structure at six regions of interest (ROI) in both forelimbs. The coefficient of variation was <3%. The structures were compared between each other and the values of each structure at the ROIs. Age, height at the withers, body mass index, diameter of the Os metacarpale III and the circumference of the mid-metacarpal region showed significant influences on some of the parameters. Reference values for the tendons and ligaments in the palmar metacarpal region in sound Haflinger mares were obtained and the influence of different factors was tested. Description of the dimensional relationship between the structures and comparison of the data with the literature provide knowledge required for adequate veterinary care.

Equine αS1-casein: Characterization of alternative splicing isoforms and determination of phosphorylation levels

αS1-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's αS1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of αS1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine αS1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped.

Two-dimensional cartography of equine β-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction.

The primary structure of a low‐Mr multiphosphorylated variant of β‐casein in equine milk

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine beta-casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.

Severe acute inflammatory reaction (SAIR) of the fetlock joint after intraarticular hyaluronate injection in a horse

Hyaluronate (HA) was administered by intra-articular injection to a 13-year-old Haflinger mare for treatment of metacarpophalangeal osteoarthritis. Ten hours after the injection, a severe inflammatory reaction developed in the treated joint. While awaiting results of synovial fluid analysis, treatment for iatrogenic infectious arthritis was initiated, but the analysis did not confirm sepsis. Clinical signs improved significantly following systemic non-steroidal anti-inflammatory medication and the horse was discharged three days later. Following an intravenous hyaluronate injection, four days after discharge, the synovitis recurred. Synovial fluid analysis did not show any abnormalities, but the clinical signs were severe. The severe acute inflammatory reaction required systemic non-steroidal anti-inflammatory and intra-articular corticosteroid treatment in order to resolve the problem.

One year old fillies can be successfully used as embryo donors

One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12–16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF 2α at day 25. Fillies’ embryo recovery rate and their recipients’ pregnancy rate at day 25 were compared with those achieved in two years old and mature mares of the same breed, subjected to the same management. Embryo recovery rate was 21/44 (47.7%), 12/16 (75%) and 22/26 (84.6%) ( P > 0.01) for one year old, two years old and mature mares, respectively. Five/7 (71.4%) one year old donors’ embryos resulted in a pregnancy after transfer and 4/7 (57.1%) developed until day 25. Significant differences in pregnancy rates after transfer between donors’ age groups were not observed; no short term side effects resulted from the use of fillies as embryo donors. This study showed that one year old mares employed as embryo donors produce embryos both morphologically normal and able to develop in recipient mares at least up to day 25 of pregnancy.

Determination of the phosphorylation level and deamidation susceptibility of equine β-casein

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.