Organogenesis and foetal haemodynamics during the normal gestation of healthy black‐rumped agoutis (Dasyprocta prymnolopha,Wagler, 1831) bred in captivity

The objective of this study was to define the patterns of organogenesis and foetal haemodynamics during the normal gestation of healthy agoutis (Dasyprocta prymnolopha) kept in captivity. Thirty pregnant agoutis that ranged in size from small to medium and weighed between 2.5 and 3 kg underwent B‐mode and Doppler ultrasonography for the biometric evaluation of the foetal organs. The foetal aortic blood flow proved to be predominantly systolic, and the measured flow velocity was 78.89 ± 2.95 cm/s, with a maximum pressure gradient of 2.12 ± 0.27 mmHg. The liver was characterized by its large volume, occupying the entire cranial aspect of the abdominal cavity, and it was associated cranially with the diaphragm and caudally with the stomach. The flow velocity in the portal vein was estimated to equal 12.17 ± 2.37 cm/s, with a resistivity index of 0.82 ± 0.05. The gallbladder was centrally located and protruded cranially towards the diaphragm. The spleen was visualized as an elongated structure with tapered cranial and caudal extremities, and the foetal kidneys were visualized bilaterally in the retroperitoneal region, with the right kidney positioned slightly more cranially than the left. The morphological characterization and hemodynamic analysis of the foetal organs of black‐rumped agoutis via B‐mode and Doppler ultrasonography allow determination of the vascular network and of reference values for the blood flow required for perfusing the anatomical elements essential for maintaining the viability of foetuses at different gestational ages.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Lactose Synthase Components in Milk: Concentrations of α-Lactalbumin and β1,4-Galactosyltransferase in Milk of Cows from Several Breeds at Various Stages of Lactation

It is believed that milk production is determined by the number and activity of mammary secretory cells. Secretory activity, as assessed by milk volume, depends on secretion of the major osmole in milk, lactose, which is produced by lactose synthase. The amount of either of the two proteins in lactose synthase may regulate milk production. The objective of this study was to determine whether the concentrations in milk of the two components of lactose synthase, α‐lactalbumin (α‐LA) and β1,4‐galactosyltransferase (B4GALT), were related to genetic background, stage of lactation, breed or parity of dairy cows. α‐Lactalbumin and B4GALT concentrations were measured by ELISA and by enzyme assays, respectively, from single milk samples. Two herds with a total of 279 cows were used in the analysis. One herd contained Ayrshire, Brown Swiss, Holstein and Jersey cows; the second herd contained two groups of cows; Holsteins selected for high milk production and Holsteins with 1960s genetics. The α‐LA concentration in milk was greater in Jerseys and Ayrshires than in Holsteins and Brown Swiss. However, no difference in α‐LA concentration was observed in milk from high and low genetic merit cows in the Minnesota herd or among different genetic backgrounds in the Illinois herd. β1,4‐Galactosyltransferase concentrations were similar for all groups that were analyzed. α‐Lactalbumin concentrations were positively correlated with milk protein concentration, milk fat concentration and lactose concentration. β1,4‐Galactosyltransferase concentration in milk exhibited a strong positive correlation with number of days in milk. Although the concentration of B4GALT increased as lactation progressed, the values did not show any correlation with persistency of lactation or late lactation milk production. In conclusion, this survey shows that the two components of lactose synthase are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.     

Minimally invasive approach to lumbosacral decompression in a cadaveric canine model

AIMS: To determine efficacy of a minimally invasive (MI) surgical approach using a human MI lumbar retractor for canine lumbosacral dorsal laminectomy and partial discectomy and to compare this technique to the standard open surgical (OS) approach.

METHODS: Lumbosacral dorsal laminectomy and partial discectomy was performed on 16 large-breed canine cadavers using either a standard OS (n=8) or MI (n=8) approach. Skin and fascial incision length, procedure time, and intraoperative complications were recorded. Postoperatively specimens were evaluated for laminectomy and discectomy dimensions, and visible damage to the cauda equina and exiting nerve roots.

RESULTS: Median length of skin and fascial incisions in the OS group were longer than in the MI group (p<0.001). Median laminectomy length was similar between both approaches (p=0.234) but width was greater for the MI than OS approach (p=0.002). Both approaches achieved similar partial discectomy width (p=0.279). Overall surgical time was longer for MI approaches compared to OS, with a median of 18.5 (min 15.5, max 21.8) minutes for MI compared to 14.6 (min 13.1, max 16.9) minutes for OS (p=0.001).

CONCLUSIONS: The MI approach reduced incision lengths while retaining comparable laminectomy and discectomy dimensions. For this in vitro model the MI approach required more time to complete, but this difference may not be relevant in clinical cases.

CLINICAL RELEVANCE: Dogs undergoing lumbosacral dorsal laminectomy are commonly large-breed dogs. The traditional open approach requires a large skin incision and soft tissue dissection, especially in overweight animals. A MI approach accomplishing the same surgical result while minimising soft tissue trauma could reduce post-operative pain and recovery time, and may lower wound-related complications. Clinical studies are needed to confirm postoperative benefit and assess operating times in vivo.     

Expression and Localization of Vascular Endothelial Growth Factor and its Receptors in the Corpus Luteum During Oestrous Cycle in Water Buffaloes (Bubalus bubalis)

The aim of this study was to document the expression and localization of VEGF system comprising of VEGF isoforms (VEGF 120, VEGF 164 and VEGF 188) and their receptors (VEGFR1 and VEGFR2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors. In general, all the components of VEGF system (the VEGF isoforms and their receptors) were found in the water buffalo CL during the oestrous cycle. The mRNA as well as protein expression of VEGF system was highest during the early and mid‐luteal phase, which later steadily decreased (p < 0.05) after day 10 to reach the lowest level in regressed CL. As demonstrated by immunohistochemistry, VEGF protein was localized predominantly in luteal cells; however, VEGFR1 and VEGFR2 were localized in luteal cells as well as in endothelial cells. In conclusion, the dynamics of expression and localization of VEGF system in buffalo corpora lutea during the luteal phase were demonstrated in this study, indicating the possible role of VEGF system in the regulation of luteal angiogenesis and proliferation of luteal as well as endothelial cells through their non‐angiogenic function.     

Failure of a single postpartum prostaglandin treatment to improve the reproductive performance of dairy cows

A study was conducted to evaluate the effects of postpartum prostaglandin treatment on reproduction in 3 seasonal calving dairy herds. Recently calved lactating dairy cows were paired on herd, age, calving date and previous production index. One cow in each of the 196 pairs received a single intramuscular injection of 25 mg of the prostaglandin analogue, dinoprost, between 14 and 28 days after calving. Subsequent reproduction was monitored. Within each herd and overall, there was no significant effect of treatment on the intervals from calving to first service, mating start date to first service, calving to conception, mating start date to conception and first service to conception. Treatment also had no significant effect on 21-day submission and pregnancy rates, on the proportion of each group not pregnant at the end of mating, and on first service pregnancy rates. Responses to treatment did not vary between cows calving within 50 days of mating start date and earlier calving cows or between cows aged less than 5 years and older cows.     

Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.     

Stratification,Blinding and Placebo Effect in a Randomized,Double Blind Placebo-controlled Clinical Trial of Gold Bead Implantation in Dogs with Hip Dysplasia

The purpose of this study was to investigate the need for and choice of stratification factors, and the effects of blinding and placebo in a clinical experiment. Eighty dogs with canine hip dysplasia (CHD) were included in a randomized, placebo-controlled and double blind clinical trial with stratified parallel group design, in which body weight and degree of CHD were used as stratification factors. Thirty-eight dogs were allocated to gold bead implantation and 42 to placebo. After six months, 33 of the 42 placebo-treated dogs received gold bead implantation in an open study lasting a further 18 months. The main outcome variable in the study was change in pain signs of CHD as assessed by the owner. No significant difference in the main outcome variable, regardless of the treatment given, could be detected in the two chosen stratification factors. The only factor to influence the main outcome variable significantly was age. The blinding procedure used in the study, in which 60% of the owners correctly guessed the treatment given, was found sufficient. Of those who guessed the treatment erroneously, 88% believed the treatment given was gold bead implantation. The treatment efficacy after six months in the blinded treatment group was found to be significantly larger compared to the efficacy obtained in the open study. A significant placebo effect was therefore detected. Conclusion and Clinical Relevance: The age of the dogs influenced the outcome of the CHD treatment, and is recommended as a stratification factor. A significant placebo effect has to be expected and an optimal blinding procedure is necessary in similar clinical studies.