The membrane‐type estrogen receptor G‐protein‐coupled estrogen receptor suppresses lipopolysaccharide‐induced interleukin 6 via inhibition of nuclear factor‐kappa B pathway in murine macrophage cells

The female sex hormone estrogen exerts anti‐inflammatory effects. The G‐protein‐coupled estrogen receptor (GPER) has been recently identified as a novel membrane‐type estrogen receptor that can mediate non‐genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha‐induced expression of interleukin 6 (IL‐6) through repression of nuclear factor‐kappa B (NF‐κB) promoter activity using human breast cancer cells. Although several reports have indicated that GPER suppresses Toll‐like receptor‐induced inflammatory cytokine expression in macrophages, the molecular mechanisms of the inhibition of cytokine production via GPER remain poorly understood. In the present study, we examined GPER‐mediated inhibition of IL‐6 expression induced by lipopolysaccharide (LPS) stimulation in a mouse macrophage cell line. We found that the GPER agonist G‐1 inhibited LPS‐induced IL‐6 expression in macrophage cells, and this inhibition was due to the repression of NF‐κB promoter activity by GPER. G‐1 treatment also decreased the phosphorylation of inhibitor of κB kinases. Among the mitogen‐activated protein kinases, the phosphorylation of c‐jun N‐terminal kinase (JNK) was increased by G‐1. These findings delineate the novel mechanism of the inhibition of LPS‐induced IL‐6 through GPER‐activated JNK‐mediated negative regulation of the NF‐κB pathway in murine macrophage cells, which links anti‐inflammatory effects to estrogen.     

N‐Acetyl‐d‐galactosamine prevents soya bean agglutinin‐induced intestinal barrier dysfunction in intestinal porcine epithelial cells

Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10^?4, 0.25 × 10^?4, 0.5 × 10^?4, 1.0 × 10^?4 and 2.0 × 10^?4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.     

Combining ultrasound and lactobacilli treatment for high‐fat‐diet‐induced obesity in mice

Chronic systemic lipopolysaccharide‐induced inflammation can cause obesity. In animal experiments, lactobacilli have been shown to inhibit obesity by modifying the gut microbiota, controlling inflammation and influencing the associated gene expression. A previous study found that high‐fat‐diet‐induced (HFD) obesity was suppressed by lactobacilli ingestion in rats via the inhibition of parasympathetic nerve activity. This study explored the combined use of lactobacilli ingestion and ultrasound (US) to control body weight and body fat deposition in HFD mice over an 8‐week experimental period. Male C57BL/6J mice received an HFD during treatment and were randomly divided into four groups: (i) control group (H), (ii) lactobacilli alone (HB), (iii) US alone (HU) and (iv) lactobacilli combined with US (HUB). The US was targeted at the inguinal portion of the epididymal fat pad on the right side. At the 8th week, body weight had decreased significantly in the HUB group (15.56 ± 1.18%, mean ± SD) group compared with the HU (26.63 ± 0.96%) and H (32.62 ± 5.03%) groups (p < 0.05). High‐resolution microcomputed tomography (micro‐CT) scans revealed that the reduction in total body fat volume was significantly greater in the HUB group (69%) than in the other two experimental groups (HB, 52%; HU, 37%; p < 0.05). The reductions in the thickness of the subcutaneous epididymal fat pads were significantly greater in the HUB group (final thickness: 340 ± 7 μm) than in the H (final thickness: 1150 ± 21 μm), HB (final thickness: 1060 ± 18 μm) and HU (final thickness: 370 ± 5 μm) groups (all p < 0.05). Combination therapy with lactobacilli and US appears to enhance the reduction in body weight, total and local body fat deposition, adipocyte size and plasma lipid levels over an 8‐week period over that achieved with lactobacilli or US alone in HFD mice. These results indicate that US treatment alone can reduce hyperlipidemia in HFD mice.     

Low‐Molecular‐Weight Heparin Dosage in Newborn Foals

Background: Heparin is used in humans as prophylaxis of hypercoagulable states and disseminated intravascular coagulation (DIC). However, babies need a higher heparin dose than do adults. Septic neonate foals are at high risk of hypercoagulable state and DIC, and there is limited objective information about heparin dose for equine neonates. Objective: To assess whether neonate foals require higher dosages of low‐molecular‐weight heparin (LMWH) than adults. Animals: Eighteen healthy and 11 septic neonate foals. Methods: Experimental and clinical studies. Firstly, healthy foals were randomly distributed in 2 groups, 1 receiving 50 IU/kg SC of dalteparin and the 2nd group receiving 100 IU/kg SC of dalteparin, once daily for 3 days. Blood samples were collected before and 3, 6, 27, and 51 hours after the 1st LMWH administration. Plasma antifactor‐Xa activity was measured, together with hemostatic and hematologic parameters used to assess the risk of bleeding. Subsequently, septic foals were treated blindly either with placebo (saline) or 100 IU/kg of dalteparin for 3 days. Plasma antifactor‐Xa activity and other hemostatic parameters were determined before and after treatment. Results: Plasma antifactor‐Xa activity in healthy foals was below prophylactic activity when using the adult dosage (50 IU/kg), whereas prophylactic activities were achieved when using the double dosage (100 IU/kg). No hemorrhagic events and erythrocyte‐related complications were observed with either dosage. In the clinical study, only 4/6 septic foals had plasma antifactor‐Xa activity adequate for prophylaxis. Conclusions and Clinical Importance: Equine neonates require higher dosages of LMWH compared with adults to reach prophylactic heparinemia.     

Pharmacokinetics and bioavailability after intramuscular injection of the 5‐HT1A serotonin agonist R‐8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) in domestic goats (Capra aegagrus hircus)

To determine the bioavailability and pharmacokinetic properties of the serotonin 5‐HT_1A receptor agonist R‐8‐OH‐DPAT in goats, and 0.1 mg kg^?1 R‐8‐OH‐DPAT hydrobromide was administered intramuscularly (i.m.) and intravenously (i.v.) to six goats in a two‐phase cross‐over design experiment. Venous blood samples were collected from the jugular vein 2, 5, 10, 15, 20, 30, 40 and 60 min following treatment and analysed by liquid chromatography tandem mass spectrometry. Bioavailability and pharmacokinetic parameters were determined by a one‐compartment analysis. Mean bioavailability of R‐8‐OH‐DPAT when injected i.m. was 66%. The mean volume of distribution in the central compartment was 1.47 L kg^?1. The mean plasma body clearance was 0.056 L kg^?1 min^?1. All goats injected i.v. and two of six goats injected i.m. showed signs of serotonin toxicity. In conclusion, R‐8‐OH‐DPAT is well absorbed following i.m. injection and the observed pharmacokinetics suggest that administration via dart is feasible. Administration of R‐8‐OH‐DPAT hydrobromide, at a dosage of 0.1 mg kg^?1, resulted in the observation of clinical signs of serotonin toxicity in the goats. It is suggested that dosages for the clinical use of the compound should be lower in order to achieve the desired clinical effect without causing serotonin toxicity.     

Multiple‐Aetiology Enteric Infections Involving Non‐O157 Shiga Toxin‐Producing Escherichia coli – FoodNet, 2001–2010

We describe multiple‐aetiology infections involving non‐O157 Shiga toxin‐producing Escherichia coli (STEC) identified through laboratory‐based surveillance in nine FoodNet sites from 2001 to 2010. A multiple‐aetiology infection (MEI) was defined as isolation of non‐O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non‐O157 STEC serogroup from the same person within a 7‐day period. We compared exposures of patients with MEI during 2001–2010 with those of patients with single‐aetiology non‐O157 STEC infections (SEI) during 2008–2009 and with those of the FoodNet population from a survey conducted during 2006–2007. In total, 1870 non‐O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non‐O157 STEC; and eight involved >1 serogroup of non‐O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non‐outbreak‐associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.     

Analysis of glycosuria in okapi (Okapia johnstoni): Examination of urinary N‐acetyl‐β‐D‐glucosaminidase

We analyzed the urinary excretion of glucose and N‐acetyl‐β‐D‐glucosaminidase (NAG) in six okapis (Okapia johnstoni) in captivity to investigate the cause of their urinary sugar excretion. The urinary glucose‐positive okapi had significantly higher urinary NAG indices than the urinary glucose‐negative okapi. There was also a positive correlation between urinary glucose levels and urinary NAG indices. These results suggest that the proximal tubular function of the glycosuric okapi may have been obstructed, which impaired glucose reabsorption.     

Non‐immunosuppressive FTY720‐derivative OSU‐2S mediates reactive oxygen species‐mediated cytotoxicity in canine B‐cell lymphoma

OSU‐2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti‐tumour activity in several haematological and solid tumour models. We have recently shown OSU‐2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre‐clinical activity of OSU‐2S in spontaneous B‐cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU‐2S mediated apoptosis in canine B‐cell lines and primary B‐cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU‐2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU‐2S‐mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU‐2S as small molecule for treating people and dogs with lymphoma.     

The effect of Epigallocatechin‐3‐gallate on small intestinal morphology,antioxidant capacity and anti‐inflammatory effect in heat‐stressed broilers

This study was to investigate the effects of Epigallocatechin‐3‐gallate (EGCG) on intestinal morphology, antioxidant capacity and anti‐inflammatory response in heat‐stressed broiler. A total of 192 2‐week‐old Arbour Acres broilers chickens were divided into four groups with six replicates per group and eight chickens per replicate: one thermoneutral control group (28°C, group TN), which was fed the basal diet; and three cyclic high‐temperature groups (35°C from 7:00 to 19:00 hr; 28°C from 19:00 hr to 7:00 hr, heat stress group), which were fed the basal diet supplementation with EGCG 0 mg/kg (group HS0), 300 mg/kg (group HS300) and 600 mg/kg (group HS600). The gut morphology and intestinal mucosal oxidative stress indicators, as well as intestinal barrier‐related gene expression, were analysed. The results showed that compared with group TN, heat stress reduced the villus height (VH), activities of glutathione peroxidase (GSH‐Px), superoxide dismutase (SOD)and catalase (CAT), increased the crypt depth (CD) and malondialdehyde (MDA)content at 21, 28 and 35 days (p < 0.05). After the heat‐stressed broilers were supplemented with EGCG, VH, VH/CD (V/C), and the activities of GSH‐Px, SOD and CAT were increased, and CD and MDA content were reduced compared with those in group HS0 without EGCG supplementation at 21, 28 and 35 days (p < 0.05). The EGCG supplementation promoted the gene expression of nuclear factor‐erythroid 2‐related factor 2 (Nrf2), Claudin‐1, Mucin 2 (Muc2) and alleviated the nuclear factor‐kappa B (NF‐κB) and lipopolysaccharide‐induced tumour necrosis factor (LITAF) gene expression compared with group HS0 (p < 0.05). Moreover, intestinal morphology was strongly correlated with antioxidant ability and inflammatory response. In conclusion, EGCG alleviated the gut oxidative injury of heat‐stressed broilers by enhancing antioxidant capacity and inhibiting inflammatory response.     

Insulin‐independent actions of glucagon‐like peptide‐1 in wethers

Insulin‐independent actions of glucagon‐like peptide‐1 (GLP‐1) are not yet clear in ruminants. Four Suffolk mature wethers (60.0 ± 6.7 kg body weight (BW)) were intravenously infused with insulin (0.5 mU/kg BW/min; from 0 to 90 min) and GLP‐1 (0.5 μg/kg BW/min; from 60 to 150 min) with both hormones co‐administered from 60 to 90 min, in a repeated‐measure design under euglycemic clamp for 150 min, to investigate whether GLP‐1 has insulin‐independent actions. Jugular blood samples were taken at 15‐min intervals for plasma hormones and metabolites analysis. Compared to baseline concentrations (at 0 min), insulin infusion decreased (P < 0.05) plasma concentrations of glucagon, non‐esterified fatty acids (NEFA), lactate, nonessential amino acids (NEAA), branched‐chain amino acids (BCAA), total amino acids (TAA) and urea nitrogen (UN). Insulin plus GLP‐1 infusion induced a greater increase (P < 0.05) in plasma concentrations of insulin and triglyceride (TG), but decreased (P < 0.05) glucagon, total cholesterol (T‐Cho), NEAA and UN plasma concentrations. GLP‐1 infusion increased (P < 0.05) NEFA, β‐hydroxybutyrate and TG, but decreased (P < 0.05) glucagon, T‐Cho, NEAA, BCAA and UN plasma concentrations. In conclusion, GLP‐1 exerts extrapancreatic roles in ruminants not only insulin‐independent but probably, in contrast to non‐ruminants, antagonistic to insulin effects.     

Genetic background of CTX‐M‐15‐producing Enterobacter hormaechei ST114 and Citrobacter freundii ST265 co‐infecting a free‐living green turtle (Chelonia mydas)

CTX‐M‐type extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae have become identified in marine ecosystem constituting a serious ecological issue. In this respect, although contamination of coastal waters and seafood, and even colonization of seabirds and fishes have been increasingly reported, molecular data are lacking to elucidate the clinical impact of ESBL producers in infected marine animals. In this study, using a genomic approach, we have analysed the genetic background of CTX‐M‐15‐producing Enterobacter hormaechei (belonging to the international human clone ST114) and Citrobacter freundii (ST265) co‐infecting a free‐living green turtle (Chelonia mydas) suffering from septic arthritis, which progressed to generalized coelomitis and death. Wide resistome of these pathogens contributed to treatment failure and death of the animal.     

COX‐2 and c‐kit expression in canine gliomas

Gliomas are among the most common primary neural tumours of dogs. Cyclooxygenase‐2 (COX‐2) and c‐kit overexpression are associated with increased aggressiveness of gliomas and decreased survival in human beings. COX‐2 is the inducible form of cyclooxygenase, which catalyzes prostaglandin formation and may increase tumour proliferation and angiogenesis. C‐kit is a tyrosine kinase receptor involved in normal cell physiology; c‐kit is upregulated in some canine tumours. In this retrospective study, 20 canine gliomas were identified: 11 (55%) oligodendrogliomas, including 1 anaplastic variant; 1 (5%) oligoastrocytoma; and 8 (40%) astrocytomas, of which 2 were glioblastoma multiforme. None of the gliomas expressed COX‐2. None of the gliomas were immunoreactive for c‐kit, although all three high‐grade tumours had intramural vascular expression. Consequently, COX‐2 inhibitors would likely be ineffective against canine gliomas. C‐kit inhibitors may have an anti‐angiogenic effect in high‐grade gliomas, but would likely be ineffective in low‐ and medium‐grade tumours.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Anti‐allergic effects of Lactobacillus crispatus KT‐11 strain on ovalbumin‐sensitized BALB/c mice

In this study, we investigated the effects of oral ingestion of Lactobacillus crispatus KT‐11 strain (KT‐11) on the immune response in an allergic rhinitis mouse model, ovalbumin (OVA)‐sensitized BALB/c mice. Sneezing activity in mice that were administered a KT‐11‐supplemented diet was significantly lower than that in mice administered a KT‐11‐free diet (control diet) at age 11 weeks. We found that serum OVA‐specific immunoglobulin E (IgE) levels and total number of interleukin (IL)‐4⁺CD4⁺ spleen cells in mice that were administered a KT‐11‐supplemented diet were significantly lower than in mice administered a control diet. The ratio of spleen interferon‐γ⁺CD4⁺/IL‐4⁺CD4⁺ cells was higher in the mice administered the KT‐11‐supplemented diet compared to that in mice administered the control or L. rhamnosus GG‐supplemented diet. In contrast, the number of CD11b⁺CD80⁺ and FcεRIα⁺CD117⁺ cells was significantly lower in mice administered the KT‐11‐supplemented diet. These results suggested that KT‐11 reduced OVA‐induced allergic symptoms in BALB/c mice via the adjustment of the T helper type 1/T helper type 2 balance, and a decrease in the number of antigen‐presenting cells and high affinity IgE receptor‐positive mast cells.     

Alternate‐day fasting diet improves fructose‐induced insulin resistance in mice

Increased fructose consumption is linked to insulin resistance, weight gain, hyperlipidemia and hypertension. Although the advantages of several dietary restriction regimens have been demonstrated, the effects of alternate‐day fasting (ADF) on fructose‐induced insulin resistance have not yet been studied. This study is based on a new modification on ADF by combining the fructose‐rich solution (10% w/v) and regular mice diet. Mice were randomly allocated into four groups: ADF50% (50% restriction in chow food intake but ad libitum fructose drink), ADF100% (100% restriction for chow food but ad libitum fructose drink), control (ad libitum chow food intake plus tap water) and daily food and fructose (DFF) (had free access to both chow and fructose solution). Biweekly fasting blood sugar (FBS), glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted. All groups gained weight during the study (p < 0.05). Body weights of DFF and control groups did not differ from that of ADF groups, but ADF50% gained more (p < 0.01) weights than ADF100% through the study. Total calorie intake (feed + fast days) of ADF50% was higher than that of ADF100% (p < 0.001) and control (p < 0.03). In addition, ADF groups consumed more energy than the control and DFF groups in feed (ad libitum) days (p < 0.05). At the end of the study, the mean FBS levels in the control and ADF100% groups were similar and significantly lower in relation to that of DFF and ADF50% groups (p < 0.01). Measurements of area under the curve in GTT and ITT revealed that the ADF100% group was more insulin‐sensitive than the DFF and ADF50% groups. In conclusion, these data suggest that the ADF100% improves fructose‐induced insulin resistance in mice.     

MULTIDETECTOR‐ROW COMPUTED TOMOGRAPHY PATTERNS OF BRONCHOESPHAGEAL ARTERY HYPERTROPHY AND SYSTEMIC‐TO‐PULMONARY FISTULA IN DOGS

Anomalies involving arterial branches in the lungs are one of the causes of hemoptysis in humans and dogs. Congenital and acquired patterns of bronchoesophageal artery hypertrophy have been reported in humans based on CT characteristics. The purpose of this retrospective study was to describe clinical, echocardiographic, and multidetector computed tomography features of bronchoesophageal artery hypertrophy and systemic‐to‐pulmonary arterial communications in a sample of 14 dogs. Two main vascular patterns were identified in dogs that resembled congenital and acquired conditions reported in humans. Pattern 1 appeared as an aberrant origin of the right bronchoesophageal artery, normal origin of the left one, and enlargement of both the bronchial and esophageal branches that formed a dense network terminating in a pulmonary artery through an orifice. Pattern 2 appeared as a normal origin of both right and left bronchoesophageal arteries, with an enlarged and tortuous course along the bronchi to the periphery of the lung, where they communicated with subsegmental pulmonary arteries. Dogs having Pattern 1 also had paraesophageal and esophageal varices, with the latter being confirmed by videoendoscopy examination. Authors conclude that dogs with Pattern 1 should be differentiated from dogs with other congenital vascular systemic‐to‐pulmonary connections. Dogs having Pattern 2 should be evaluated for underlying pleural or pulmonary diseases. Bronchoesophageal artery hypertrophy can be accompanied by esophageal venous engorgement and should be included in the differential diagnosis for esophageal and paraesophageal varices in dogs.     

Comparative proteomic analysis of high‐ and low‐fertile buffalo bull spermatozoa for identification of fertility‐associated proteins

The present study identified few potential proteins in the spermatozoa of buffalo bulls that can be used as an aid in fertility determination through comparative proteomics. The sperm proteome of high‐fertile buffalo bulls was compared with that of low‐fertile buffalo bulls using two‐dimensional difference gel electrophoresis (2D‐DIGE), and the differentially expressed proteins were identified through mass spectrometric method. The protein interaction network and the functional bioinformatics analysis of differentially expressed proteins were also carried out. In the spermatozoa of high‐fertile bulls, 10 proteins were found overexpressed and 15 proteins were underexpressed at the level of twofold or more (p ≤ 0.05). The proteins overexpressed in high‐fertile spermatozoa were PDZD8, GTF2F2, ZNF397, KIZ, LOH12CR1, ACRBP, PRSS37, CYP11B2, F13A1 and SPO11, whereas those overexpressed in low‐fertile spermatozoa were MT1A, ATP5F1, CS, TCRB, PRODH2, HARS, IDH3A, SRPK3, Uncharacterized protein C9orf9 homolog isoform X4, TUBB2B, GPR4, PMP2, CTSL1, TPPP2 and EGFL6. The differential expression ranged from 2.0‐ to 6.1‐fold between the two groups, where CYP11B2 was high abundant in high‐fertile spermatozoa and MT1A was highly abundant in low‐fertile spermatozoa. Most of the proteins overexpressed in low‐fertile spermatozoa were related to energy metabolism and capacitation factors, pointing out the possible role of pre‐mature capacitation and cryo‐damages in reducing the fertility of cryopreserved buffalo spermatozoa.