Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.     

Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen Cryopreservation Improves Sperm Viability but Results in Low Fertility in Mares

The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10⁶ spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC‐treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC‐treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC‐treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S‐MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S‐MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.     

Distinct Effects of Boar Seminal Plasma Fractions Exhibiting Different Protein Profiles on the Functionality of Highly Diluted Boar Spermatozoa

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 × 10⁶ spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4–6 contained low numbers of spermatozoa (<500 × 10⁶/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1–3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1–3 appear to promote sperm survival, whereas fractions 4–6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.     

Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation,fertilization and development

This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 μM) or cilostamide (20 μM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.     

Production of Oocytes of Nile Tilapia (Oreochromis niloticus) for In Vitro Fertilization via Hormonal Treatments

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin‐releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post‐hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.     

Muscle strain injuries of the hindlimb in eight horses: diagnostic imaging,management and outcomes

Objective To describe the clinical presentation, ultrasound findings, management and outcome in horses with muscle tear injuries of the hindlimbs. Design Retrospective case series Procedure Medical records of eight horses were reviewed and information on signalment, history, presenting complaint, physical examination findings and further diagnostic tests were recorded. Diagnosis of muscle injury was determined by the presence of abnormal ultrasound findings, compared with the contralateral limb, and, when required, nuclear scintigraphy. Follow-up information was obtained via telephone interviews with owners, trainers and referring veterinarians. Results Muscle tears causing lameness were identified in the middle gluteal (3), semitendinosus (1), semimembranosus (2) and gracilis (2) muscles. Tears were classified by ultrasound imaging as partial (6) or complete (2). The degree of lameness did not appear to be indicative of the extent of injury or of completion of healing. Long-term follow-up was available for seven horses and the outcome was favourable in six cases. Conclusions and clinical relevance Ultrasonography is useful in the diagnosis and assessment of moderate to severe muscle strain injuries. The prognosis appears to be favourable in most cases, although recurrence of injury and lameness can delay the return to athletic activity and an inferior outcome with persistent gait abnormality may occur.     

Motility Characteristics and Fertilizing Capacity of Boar Spermatozoa Stained with Hoechst 33342

Flow cytometry sorting of spermatozoa using fluorescence dye Hoechst 33342 is the only effective sex selection methodology validated in numerous laboratories. This study was carried out to determine the effect of Hoechst 33342 on the motility and fertility of stained boar spermatozoa. Experiment 1 evaluated motility parameters (percentage of motile spermatozoa, velocity, angularity and oscillation) of boar spermatozoa stained with Hoechst 33342 by a computer‐aided sperm analysis (CASA) instrument. Spermatozoa (30 million/ml) were divided into five treatment groups and stained during 1 h at 35°C with 9, 18, 27, 60 and 90 μM of H33342. There were no differences in sperm motility patterns nor percentages of motile spermatozoa incubated in the presence of 9, 18 or 27 μM. Percentage of motile spermatozoa and motility parameters decreased significantly (p < 0.05) at 60 μM of Hoechst 33342. Spermatozoa were immotile at concentration of 90 μM. In experiment 2, pregnancy rates, farrowing rates and litter size from sows (n = 275) artificially inseminated (AI) with either Hoechst 33342 stained (27 μM) or unstained (control) spermatozoa were determined. Sows inseminated with stained spermatozoa had no significant lower pregnancy rate (88.33%) as compared with controls (90.32%). Staining neither affected farrowing rates (85.0 vs 87.7%) nor total number of piglets born (10.56 ± 0.32 vs 10.47 ± 0.24, stained and controls, respectively). No phenotypical abnormalities were registered among the newborn piglets. The data suggest that incubating spermatozoa with Hoechst 33342 at levels required for X‐ and Y‐bearing chromosome sperm sorting, does not impair sperm viability or their fertility after AI.     

Factors Affecting Pregnancy Rate in Artificial Insemination with Frozen Semen During Non-Breeding Season in Murciano–Granadina Goats: a Field Assay

An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano-Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.