Functional proteomic analysis of crossbred (Holstein Friesian × Sahiwal) bull spermatozoa

Male infertility is one of the prime concerns of dairy cattle production. The study was designed to find out differentially expressed proteins in categorized crossbred (Holstein Friesian × Sahiwal) bull semen to serve as potential biomarkers for male infertility. Frozen crossbred bull semen with satisfactory phenotypic records were defined as “good” and “poor” based on their fertility rates. A total of 1,547 proteins were detected in bull spermatozoa using liquid chromatography–mass spectrometer (LC‐MS/MS) analysis. Results revealed that 558 (36.1%) and 653 (42.2%) proteins were expressed to good and poor quality bull spermatozoa, respectively. A total of 336 proteins (21.7%) were reported to be unique for both good and poor quality bull semen, and among the common proteins, 224 (66.7%) and 112 (33.3%) were up‐ and downregulated in good and poor quality categorized bull semen, respectively. Gene Ontology analysis of global proteomes identified different signalling pathways, and most of them were related to cellular motility, immune systems as well as cellular metabolisms. The distinctive presence of some of the proteins may provide an insight into the molecular mechanistic role played by these proteins in crossbred bull infertility.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Assessment of pregnancy‐associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera

Pregnancy‐associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)‐PAG systems: RIA‐1 (antiserum raised against bovine PAG67kDa; AS#497), RIA‐2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA‐3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA‐2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA‐2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA‐3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA‐1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG‐RIA systems to measure PAG concentration in swamp buffalo samples.     

Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm‐Ovis‐Halomax during in vitro capacitation of cryopreserved ram spermatozoa

This study aimed to compare the ability of sperm chromatin structure assay (SCSA^®) and Sperm‐Ovis‐Halomax^® to detect DNA fragmentation in frozen‐thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF‐ESS) at multiple time points (0–240 min). Incubation in SOF‐ESS had no significant effects on SCSA^® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm‐Ovis‐Halomax^® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA^® and Sperm‐Ovis‐Halomax^® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.     

A retrospective analysis of radiation therapy for the treatment of feline vaccine‐associated sarcoma*

We retrospectively evaluated predictive prognostic factors in 73 cats with vaccine‐associated sarcoma given postsurgical curative (n = 46, most with clean margins) or coarse fractionated radiotherapy (n = 27, most with either macroscopic disease or dirty margins). The former animals displayed a median survival of 43 months and a median progression free interval (PFI) of 37 months, the latter reached a median survival of 24 months and a median PFI of 10 months. In cats undergoing coarse fractionated therapy, factors predictive of a better outcome included lack of visible mass (n = 10) as opposed to macroscopic disease (n = 17, survival: 30 versus 7 months, P = 0.025; PFI: 20 versus 4 months, P = 0.01), adjuvant chemotherapy for gross disease (n = 5/17, survival: 29 versus 5 months, P = 0.04) and a smaller number of surgeries preceding radiation therapy (coeff = 0.41, P = 0.03). The Ki67 index was not predictive for survival. We concluded that postsurgical curative and coarse fractionated radiotherapy are effective legitimate options for managing vaccine‐associated sarcomas.     

Growth of rumen papillae in weaned calves is associated with lower expression of insulin‐like growth factor‐binding proteins 2, 3, and 6

This study aimed to characterize the relationship between the growth of rumen papillae in calves and the mRNA expression of insulin‐like growth factor‐binding proteins (IGFBPs) in the rumen papillae. The length of rumen papillae, the mRNA expression of IGFBPs in rumen papillae by quantitative real‐time PCR, and the presence of insulin‐like growth factors I and II (IGF‐I and II) by immunohistochemistry (IHC) were analyzed in nine Holstein calves divided into three groups: suckling (2 weeks, n = 3), milk‐continued (8 weeks, n = 3), and weaned (8 weeks, n = 3). The length of rumen papillae was greater (p < 0.01) in weaned calves than in suckling and milk‐continued calves, whereas the expressions of IGFBP2, IGFBP3, and IGFBP6 genes were lower (p < 0.05) in the rumen papillae of weaned calves than in milk‐continued calves. Thus, rumen papillae length and IGFBP2, 3, and 6 expressions were negatively correlated. The IHC analysis showed that IGF‐I and IGF‐II were present in the rumen epithelium of calves. These results suggested that the growth of rumen papillae after weaning is associated with the induction of IGFs by the low levels of IGFBP2, IGFBP3, and IGFBP6.     

Evaluation of T2‐weighted versus short‐tau inversion recovery sagittal sequences in the identification and localization of canine intervertebral disc extrusion with low‐field magnetic resonance imaging

Sagittal T2‐weighted sequences (T2‐SAG) are the foundation of spinal protocols when screening for the presence of intervertebral disc extrusion. We often utilize sagittal short‐tau inversion recovery sequences (STIR‐SAG) as an adjunctive screening series, and experience suggests that this combined approach provides superior detection rates. We hypothesized that STIR‐SAG would provide higher sensitivity than T2‐SAG in the identification and localization of intervertebral disc extrusion. We further hypothesized that the parallel evaluation of paired T2‐SAG and STIR‐SAG series would provide a higher sensitivity than could be achieved with either independent sagittal series when viewed in isolation. This retrospective diagnostic accuracy study blindly reviewed T2‐SAG and STIR‐SAG sequences from dogs (n = 110) with surgically confirmed intervertebral disc extrusion. A consensus between two radiologists found no significant difference in sensitivity between T2‐SAG and STIR‐SAG during the identification of intervertebral disc extrusion (T2‐SAG: 92.7%, STIR‐SAG: 94.5%, P = 0.752). Nevertheless, STIR‐SAG accurately identified intervertebral disc extrusion in 66.7% of cases where the evaluation of T2‐SAG in isolation had provided a false negative diagnosis. Additionally, one radiologist found that the parallel evaluation of paired T2‐SAG and STIR‐SAG series provided a significantly higher sensitivity than T2‐SAG in isolation, during the identification of intervertebral disc extrusion (T2‐SAG: 78.2%, paired T2‐SAG, and STIR‐SAG: 90.9%, P = 0.017). A similar nonsignificant trend was observed when the consensus of both radiologists was taken into consideration (T2‐SAG: 92.7%, paired T2‐SAG, and STIR‐SAG = 97.3%, P = 0.392). We therefore conclude that STIR‐SAG is capable of identifying intervertebral disc extrusion that is inconspicuous in T2‐SAG, and that STIR‐SAG should be considered a useful adjunctive sequence during preliminary sagittal screening for intervertebral disc extrusion in low‐field magnetic resonance.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Evaluation of a multi‐agent chemotherapy protocol combining dexamethasone,melphalan, actinomycin D,and cytarabine for the treatment of resistant canine non‐Hodgkin high‐grade lymphomas: a single centre's experience

The DMAC protocol (dexamethasone, melphalan, actinomycin‐D, cytarabine) has been evaluated in American studies for the treatment of relapsed canine lymphoma, comparing similarly to other rescue protocols. The aim of this study was to evaluate efficacy and toxicity of DMAC, in a larger UK cohort of resistant canine lymphomas. Medical records of dogs with resistant non‐Hodgkin high‐grade lymphomas that received DMAC as a rescue protocol were reviewed from 2007 to 2017. Response, time from initiation to discontinuation (TTD) and toxicity (Veterinary Cooperative Oncology Group criteria) were assessed. One hundred dogs were included; 86 received CEOP (modified CHOP including epirubicin) as first‐line treatment. Thirty‐five dogs (35%) responded: 21 complete responders (CRs) and 14 partial responders (PRs). Responders had significantly longer TTD (P < 0.001) compared with non‐responders: 62 days (range 28‐952) for CR vs 32 days (range 20‐70) for PR. Six CR received more than six cycles of DMAC (range 7‐36 cycles) and experienced a longer TTD (median 508, range 126‐952 days). Thrombocytopenia occurred in 45% (24 grade 1‐2, 21 grade 3‐4) and neutropenia in 36% of cases (29 grade 1‐2, 7 grade 3‐4). Gastrointestinal toxicity occurred in 42% of dogs (40 grade 1‐2, 2 grade 3‐4). Owing to chemotherapy toxicity, treatment was discontinued in five, and hospitalization required in six cases. In this study, response to DMAC was lower and of generally shorter duration than previously reported. Toxicity was high, but infrequently led to hospitalization or discontinuation of treatment.