Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.     

Linkage disequilibrium and effective population size in Gir cattle selected for yearling weight

Linkage disequilibrium (LD) plays an important role in genomic selection and mapping of quantitative trait loci (QTL). This study investigated the pattern of LD and effective population size (N_e) in Gir cattle selected for yearling weight. For this purpose, 173 animals with imputed genotypes (from 18 animals genotyped with the Illumina BovineHD BeadChip and 155 animals genotyped with the Bovine LDv4 panel) were analysed. The LD was evaluated at distances of 25–50 kb, 50–100 kb, 100–500 kb and 0.5–1 Mb. The N_e was estimated based on 5 past generations. The r² values (a measure of LD) were, respectively, .35, .29, .18 and .032 for the distances evaluated. The LD estimates decreased with increasing distance of SNP pairs and LD persisted up to a distance of 100 kb (r² = .29). The N_e was greater in generations 4 and 5 (24 and 30 animals, respectively) and declined drastically after the last generation (12 animals). The results showed high levels of LD and low N_e, which were probably due to the loss of genetic variability as a consequence of the structure of the Gir population studied.     

Morphological changes of physeal cartilage and secondary ossification centres in the developing femur of the house mouse (Mus musculus): A micro‐CT based study

In mammals, long bones are formed by ossification of a cartilaginous mould during early stages of development, through the formation of structures called the primary ossification centre, the secondary ossification centres (SOCs) and the physeal cartilages (PCs). The PC is responsible for long bone growth. The morphology of the PC and the SOCs varies during different stages of femoral growth. In this respect, several details involving the process of murine femoral development are lacking. In the present study, a morphological characterization of femur development from the embryonic period to adulthood in mice was studied using micro‐computed tomography (micro‐CT). To achieve this aim, femora were collected at embryonic day (E) 14.5, E16.5 and E18.5 and at postnatal day (P)1, P7, P14, P35, P46 and P52. CT images were obtained using a micro‐CT scanner (X‐SkyScan 1172; Micro Photonics) and analysed using the micro‐CT 3D visualization software Mimics (Materialise NV, Leuven, Belgium) and NRecon (Micro Photonics). The results of the present study revealed that the femur and its PCs and SOCs undergo morphological changes during different stages of development, including changes in their shape as well as position and thickness. These changes may be due to the response of the femur to mechanical loads imposed by muscle surrounding the bone during these stages of development. The result of the present study is important to improve our knowledge related to ossification and growth patterns of mouse femur during development.     

Abnormal Sysmex XT‐2000iV DIFF scattergram in a cat with a prominent mastocytemia

A 2‐year‐old neutered male domestic shorthair cat was presented to the emergency service of the National Veterinary School of Toulouse (France) for acute vomiting and diarrhea with lethargy, inappetence, and adypsia for the past 48 hours. Complete blood counts were performed with the ProCyte DX at the emergency department and with the Sysmex XT‐2000iV at the laboratory 2 weeks later. The scattergrams from the two analyzers revealed similar unusual and abnormal dot plots. The Sysmex XT‐2000iV DIFF scattergram also showed no clear separation between different leukocyte populations. The eosinophil cluster was in an abnormal location compared with that of the “typical” location in a normal cat. A blood smear evaluation revealed the presence of numerous mast cells. Thus, we hypothesized that the Sysmex XT‐2000iV had detected the mast cell population, and this led to errors in the differential counts. To explore this hypothesis, we manually gated on the DIFF scattergram and performed a manual differential on the blood smear. With this new gating strategy, the Sysmex XT‐2000iV and manual differentials were similar. Thus, in the case of systemic mastocytosis, mast cells can be located between the lymphocyte, monocyte, and eosinophil clusters on scattergrams.