Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.

Methods

Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 μg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000).

Result(s)

Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P < 0.05) in samples added with dilutions of rabbit antiserum lower than 1:80,000 (one foetus) or 1:10,000 (four foetuses). It was also shown that the recovery of added bPL (12 ng/mL) was markedly reduced in those samples in which exogenous rabbit anti-bPL were added at dilutions lower than 1:20,000. Concentrations of foetal bPL were determined in samples from cannulated foetuses. In foetuses 1 and 6, bPL concentrations remained almost unchanged (<5 ng/mL) during the whole experimental period. In Foetus 3, bPL concentrations decreased immediately after IgG infusion and thereafter, they increased until parturition.

Conclusion(s)

The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.     

Bovine thyroglobulin gene polymorphisms and their association with sexual precocity in Guzerat bulls

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well‐described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.     

Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides

Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two‐dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78‐kDa glucose‐regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta‐1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.     

Effects of buprenorphine,butorphanol or tramadol premedication on anaesthetic induction with alfaxalone in common marmosets (Callithrix jacchus)

Objective

To investigate the clinical and physiological effects of intravenous (IV) alfaxalone alone or in combination with buprenorphine, butorphanol or tramadol premedication in marmosets.

Prospective evaluation of pancarpal arthrodesis for carpal injuries in working dogs in New Zealand,using dorsal hybrid plating

Abstract

AIM: To determine whether working dogs in New Zealand with carpal injuries and treated with unilateral pancarpal arthrodesis (PCA), using a dorsal hybrid-plating method, are able to return to satisfactory working ability.

METHODS: Fourteen working dogs presented to the Veterinary Specialist Group (VSG) and the Massey University Veterinary Teaching Hospital (MUVTH) with carpal injuries were prospectively treated using dorsal hybrid plating. Dogs were eligible if actively involved in farm, hunting or police work. Dogs had a standardised PCA surgical procedure performed, and similar instructions for post-operative care were provided. Dogs were re-evaluated clinically and radiographically at 6 weeks, 6 months, and 12 months after surgery. A questionnaire was completed by 12 owners, to assess each dog's working ability.

RESULTS: Twelve months following arthrodesis, 10/12 (83%) dogs could perform most or all duties normally. Eleven owners (92%) reported that the result of the surgery met their expectations, and nine owners (75%) were very satisfied with the outcome of the surgery. No owners were disappointed or very disappointed with the surgical outcome. Post-operative complications requiring surgical removal of the implant occurred in three (25%) dogs.

CONCLUSIONS: Unilateral PCA using a standardised surgical procedure and dorsal hybrid plating of carpal injuries has a good prognosis for working dogs in New Zealand to return to work.

CLINICAL RELEVANCE: These results may allow veterinarians to provide a more accurate prognosis to owners of working dogs that have debilitating carpal injury